Methods and compositions for preparing transplant tissue

ABSTRACT

Methods and compositions for preparing and priming a tissue graft for an accelerated therapeutic effect are provided herein. In one embodiment, the method includes obtaining a tissue containing viable cells from a donor, wherein the viable cells are endogenous to the tissue and remain resident in the tissue; and priming the viable cells with one or more stimuli comprising simulated hypoxia to produce a primed tissue, wherein when grafted to a recipient the primed tissue provides a benefit compared to non-primed tissue.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a divisional of U.S. patent application Ser.No. 15/680,012, filed Aug. 17, 2017, now allowed, and claims the benefitof U.S. Provisional Application No. 62/376,843, filed Aug. 18, 2016, theentire disclosure of which is incorporated by reference herein.

FIELD

Compositions and methods disclosed herein relate to transplant tissue,in particular preparation of enhanced transplant tissue prior toclinical usage.

BACKGROUND

Transplantation of isolated tissue or whole organs is a criticaltherapeutic strategy that is widely utilized in the treatment ofpatients. Tissue transplants can be in the form of an allograft,autograft, or xenograft. An allograft transplant is the transplantationof cells, tissues, or organs to a recipient from a geneticallynon-identical recipient of the same species. An autograft is thetransplantation of tissue from one site to another on located on thesame patient. A xenograft is the transplantation of tissue from anotherspecies. In each case the tissue is removed from a donor, handled, andtransplanted to a recipient to regenerate tissue.

Known methods and procedures for tissue and organ transplantation havesignificant limitations. For example, the handling and processing oftransplant tissue may result in inflammation, a decreased biologicresponse upon transplantation, or morbidity of patients. Further, eventissues from a genetically similar donor, may contain cells that are notimmunologically compatible or reactive with the recipient.

Therefore, methods and compositions for providing improved transplanttissue are urgently needed.

SUMMARY

In one aspect, a method for preparing a primed tissue graft is provided.The method can include obtaining a tissue containing viable cells from adonor, wherein the viable cells are endogenous to the tissue and remainresident in the tissue; and priming the viable cells with one or morestimuli to produce a primed tissue graft, wherein when used to treat apatient the primed tissue graft provides a benefit compared tonon-primed tissue.

In various embodiments, the tissue is an allograft, autograft orxenograft tissue. The tissue can be obtained from any source, such asone or more of placenta, amnion, chorion, umbilical cord, Wharton'sJelly, bone, periosteum, cartilage, meniscus, spinal disc, muscle,tendon, ligament, adipose, skin, cardiovascular tissue, peritoneum,fascia, interstitial tissue such as intestinal submucosa, nerve, cornea,visceral organ, reproductive tissue, hair follicles, foreskin, anddental tissue.

In some embodiments, the viable cells are not isolated from the tissueand comprise non-terminally differentiated cells and/or differentiatedcells.

In certain embodiments, the one or more stimuli can be a transient orprolonged exposure to one or more biochemical agents, deprivation ofnutrient(s), change in oxygen level, application of mechanical stressand/or electromagnetic field, change in temperature, change in pH,irradiation, shockwave treatment, pressure level, or any combination ofthe foregoing.

In some embodiments, the biochemical agent can be one or more of agrowth-inductive component, medium component, cell death inhibitor,antioxidant, vitamin, enzyme, expression of antimicrobial,anti-inflammatory, anti-scarring, or angiogenic proteins, anddifferentiation-inducing factor such as dexamethasone and indomethacine.The change in oxygen level can be hypoxia or hyperoxia due toatmospheric oxygen levels and/or exposure to a medium ingredient thatsimulates hypoxia or hyperoxia such as deferoxamine. The mechanicalstress can be one or more of fluid flow, shear, stretch, compression,torque, static force, cyclic force and pulsatile force.

In various embodiments, the benefit provided by the primed tissue graftcomprises one or more of (1) altered cell adhesion, altered cellproliferation, altered cell survival, maintenance of cell viability,mainetenance of cell phenotype and/or altered cell migration; (2)induced cell differentiation, de-differentiation and/ortransdifferentiation; (3) production of extracellular matrix and/orbiochemical factors; (4) faster or improved healing or remodeling; (5)reduced risk of infection; (6) reduced risk of graft rejection; and (7)reduced level of inflammation.

In some embodiments, the method can further include cryopreserving theprimed tissue graft and grafting the cryopreserved tissue.Alternatively, primed tissue graft can be grafted to a recipient withoutcryopreservation.

In some embodiments, the method can further include storage of theprimed tissue graft at frozen temperatures, at refrigeratedtemperatures, or at above refrigerated temperatures.

In some embodiments, the method can further include maintaining theintegrity of the primed tissue graft.

In some embodiments, the method can further include preparing the primedtissue graft and optionally the viable cells resident in the primedtissue graft for the environment into which the primed tissue graft willbe implanted into.

In some embodiments, the method can further include grafting the primedtissue graft to the patient, wherein at the time of grafting, the primedtissue graft contains the viable cells.

In certain embodiments, immunoreactive cells can be removed prior tografting. The primed tissue graft can also be devitalized ordecellularized prior to grafting. Devitalizing can include physicaltreatment (e.g., freeze-and-thaw cycles, sonication, pressure, vacuum,and mechanical agitation), enzymatic treatment (e.g., Trypsin) and/orchemical treatment (e.g., sodium deoxycholate, Triton X solutions).

Another aspect relates to an artificially primed tissue graft,comprising: a tissue obtained from a donor; and viable cells that areendogenous to the tissue and remain resident in the tissue, wherein theviable cells have been primed with one or more stimuli to produce aprimed tissue graft, wherein when used to treat a patient the primedtissue provides a benefit compared to non-primed tissue.

A further aspect relates to an artificially primed tissue graft,comprising: a tissue obtained from a donor; and extracellular componentsproduced by viable cells that are endogenous to and resident in thetissue, wherein the viable cells have been primed with one or morestimuli to produce the extracellular components and subsequently atleast partially devitalized or decellularized, wherein the extracellularcomponents comprise extracellular matrix and/or secreted factors and areassociated with the primed tissue graft; wherein when used to treat apatient the primed tissue graft provides a benefit compared tonon-primed tissue.

Yet another aspect relates to an artificially conditioned medium,comprising: a base medium for priming viable cells that are endogenousto and resident in a tissue obtained from a donor; and one or morefactors secreted by the viable cells during priming, wherein whengrafted or administered to a recipient the conditioned medium provides abenefit.

In some embodiments, the artificially conditioned medium can furtherinclude a component for extending cell viability.

In some embodiments, the artificially conditioned medium can furtherinclude a component for pre-conditioning tissue grafts and cellsendogenous to the tissue grafts.

In a further aspect, a method for providing a primed tissue graft isprovided. The method can include obtaining a tissue containing viablecells from a donor, wherein the viable cells are endogenous to thetissue and remain resident in the tissue; and priming the tissue graftwith one or more stimuli to produce a primed tissue graft, wherein whenused to treat a patient, the primed tissue graft provides a benefitcompared to non-primed tissue.

In various embodiments, the tissue is an allograft, autograft orxenograft tissue. The tissue can be obtained from any source, such asone or more of placenta, amnion, chorion, umbilical cord, Wharton'sJelly, bone, periosteum, cartilage, meniscus, spinal disc, muscle,tendon, ligament, adipose, skin, cardiovascular tissue, peritoneum,fascia, interstitial tissue such as intestinal submucosa, nerve, cornea,visceral organ, reproductive tissue, hair follicles, foreskin, anddental tissue.

In certain embodiments, the one or more stimuli can be a transient orprolonged exposure to one or more biochemical agents, deprivation ofnutrient(s), change in oxygen level, application of mechanical stressand/or electromagnetic field, change in temperature, change in pH,irradiation, shockwave treatment, pressure level, or any combination ofthe foregoing.

In various embodiments, the benefit provided by the primed tissue graftcomprises one or more of (1) altered cell adhesion, altered cellproliferation, altered cell survival, maintenance of cell viability,mainetenance of cell phenotype and/or altered cell migration; (2)induced cell differentiation, de-differentiation and/ortransdifferentiation; (3) production of extracellular matrix and/orbiochemical factors; (4) faster or improved healing or remodeling; (5)reduced risk of infection; (6) reduced risk of graft rejection; and (7)reduced level of inflammation.

In some embodiments, the method can further include grafting the primedtissue graft to the patient, wherein at the time of grafting, the primedtissue graft contains the viable cells.

Also provided herein is a composition comprising the artificially primedtissue graft disclosed herein and the artificially conditioned mediumdisclosed herein.

Another aspect relates to a method for providing a primed tissue graft,comprising: obtaining a tissue containing viable cells from a donor,wherein the viable cells are endogenous to the tissue and remainresident in the tissue; and priming the viable cells with one or morestimuli to increase cell viability. In some embodiments, the one or morestimuli include a cell death or apoptosis inhibitor.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1B illustrate a simplified schematic of cellular reprograming;

FIGS. 2A-2C illustrate osteogenic differentiation of cryopreservedamnion tissue;

FIGS. 3A-3F illustrate osteogenic differentiation of fresh minced amnionand chorion tissue;

FIGS. 4A-4F illustrate osteogenic differentiation experiments ofcryopreserved minced amnion and chorion tissue;

FIGS. 5A-5B illustrate chondrogenic differentiation of isolated amnioncells in various configurations;

FIGS. 6A-6G illustrate osteogenic differentiation of isolated amnioncells in various configurations;

FIGS. 7A-7B illustrate osteogenic differentiation of amnion in a sheetconfiguration;

FIGS. 8A-8E illustrate osteogenic and adipogenic differentiation ofamnion tissue; and

FIGS. 9A-9C illustrate osteogenic differentiation of amnion tissue.

DETAILED DESCRIPTION

The compositions and method disclosed herein relate to priming oftissues and cells before grafting, and primed tissue, primed cells andconditioned media prepared therefrom. In some embodiments, a method forproviding a primed tissue can include: obtaining a tissue containingviable cells from a donor, wherein the viable cells are endogenous tothe tissue and remain resident in the tissue; and priming the viablecells with one or more stimuli to produce a primed tissue, wherein whengrafted to a recipient the primed tissue provides a benefit compared tonon-primed tissue. The benefit can include one or more of (1) alteredcell adhesion, altered cell proliferation, altered cell survival,maintenance of cell viability, mainetenance of cell phenotype and/oraltered cell migration; (2) induced cell differentiation,de-differentiation and/or transdifferentiation; (3) production ofextracellular matrix and/or biochemical factors; (4) faster or improvedhealing or remodeling; (5) reduced risk of infection; (6) reduced riskof graft rejection; and (7) reduced level of inflammation. In variousembodiments, the viable cells are not isolated from the tissue andcomprise non-terminally differentiated cells and/or differentiatedcells.

The tissue can be an allograft, autograft or xenograft tissue. In someembodiments, the tissue is obtained from one or more of placenta,amnion, chorion, umbilical cord, Wharton's Jelly, bone, periosteum,cartilage, meniscus, spinal disc, muscle, tendon, ligament, adipose,skin, cardiovascular tissue, peritoneum, fascia, nerve, cornea, visceralorgan, reproductive tissue, hair follicles, foreskin, and dental tissue.In some embodiments, the tissue is selected from the group consisting ofan adipose tissue, an amnion tissue, an artery tissue, a bone tissue, acartilage tissue, a chorion tissue, a colon tissue, a dental tissue, adermal tissue, a duodenal tissue, an endothelial tissue, an epithelialtissue, a fascial tissue, a gastrointestinal tissue, a growth platetissue, an intervertebral disc tissue, an intestinal mucosal tissue, anintestinal serosal tissue, a ligament tissue, a liver tissue, a lungtissue, a mammary tissue, a meniscal tissue, a muscle tissue, a nervetissue, an ovarian tissue, a parenchymal organ tissue, a pericardialtissue, a periosteal tissue, a peritoneal tissue, a placental tissue, askin tissue, a spleen tissue, a stomach tissue, a synovial tissue, atendon tissue, a testes tissue, an umbilical cord tissue, a urologicaltissue, a vascular tissue, a vein tissue, and any combination thereof.

In certain embodiments, the one or more stimuli can be a biochemicalagent, deprivation of nutrient(s), change in oxygen level, mechanicalstress, electromagnetic field, change in temperature, change in pH,irradiation, shockwave treatment, pressure level, or any combination ofthe foregoing.

1. Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisdisclosure belongs.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., at least one) of the grammatical object of the article.By way of example, “an element” means one element or more than oneelement.

As used herein, the term “about” means within 20%, more preferablywithin 10% and most preferably within 5%. The term “substantially” meansmore than 50%, preferably more than 80%, and most preferably more than90% or 95%.

The term “adipokine” as used herein refers to a factor secreted byadipose tissue.

The term “adipocyte” as used herein refers to the functional cell typeof fat, or adipose tissue that is found throughout the body,particularly under the skin. Adipocytes store and synthesize fat forenergy, thermal regulation and cushioning against mechanical shock.Although the lineage of adipocytes is still unclear, it appears thatMSCs can differentiate into two types of lipoblasts, one that give riseto white adipocytes and the other to brown adipocytes. Both types ofadipocytes store fat.

The term “adipogenic” as used herein refers to a potential of precursorcells to differentiate into fat forming or adipocompetent cells, whereinthe adipogenic cells include one or more of an adipogenic stem cell, atissuegenic cell, a precursor cell, a progenitor cell, an immature cell,a non-terminally differentiated cell, a cell with differentiationpotential, or a combination thereof.

The term “adipose stem cell” (ASC) as used herein refers to pluripotentstem cells, MSCs and more committed adipose progenitors and stromaobtained from adipose tissue.

As used herein, the term “allograft” refers to a graft of tissueobtained from a donor of the same species as, but with a differentgenetic make-up from, the recipient, as a tissue transplant between twohumans. The term allograft is generally referred to as an implant.

The term “allogeneic” as used herein refers to being geneticallydifferent although belonging to or obtained from the same species.

The term “amniotic stem cells” as used herein refers to pluripotent stemcells, multipotent stem cells and progenitor cells derived from amnioticmembrane, which can give rise to a limited number of cell types in vitroand/or in vivo under an appropriate condition, and expressly includesboth amniotic epithelial cells and amniotic stromal cells. Cells foundin the amniotic fluid are derived from the amniotic mebrane and can alsobe referred to as “amniotic stem cells”.

The terms “artificial” and “artificially” as used herein refers to acomposition such as a medium that is non-naturally existing and/or isprepared in vitro using a non-naturally occurring process.

The term “autologous” as used herein means derived from the sameorganism.

The term “autologous graft” or “autograft” as used herein refers to atissue that is grafted into a new position in or on the body of the sameindividual.

The term “basic fibroblast growth factor” (bFGF) as used herein refersto a multifunctional effector for many cells of mesenchymal andneuroectodermal origin that is a potent inducer of neovascularizationand angiogenesis.

The term “biomarkers” (or “biosignatures”) as used herein refers topeptides, proteins, nucleic acids, antibodies, genes, metabolites, orany other substances used as indicators of a biologic state. It is acharacteristic that is measured objectively and evaluated as a cellularor molecular indicator of normal biologic processes, pathogenicprocesses, or pharmacologic responses to a therapeutic intervention.

The term “bone” as used herein refers to a hard connective tissueconsisting of cells embedded in a matrix of mineralized ground substanceand collagen fibers. The fibers are impregnated with a form of calciumphosphate similar to hydroxyapatite as well as with substantialquantities of carbonate, citrate and magnesium. Bone consists of a denseouter layer of compact substance or cortical substance covered by theperiosteum and an inner loose, spongy substance; the central portion ofa long bone is filled with marrow.

The term “bone morphogenetic protein (BMP)” as used herein refers to agroup of cytokines that are part of the transforming growth factor-ß(TGF-ß) superfamily. BMP ligands bind to a complex of the BMP receptortype II and a BMP receptor type I (Ia or Ib). This leads to thephosphorylation of the type I receptor that subsequently phosphorylatesthe BMP-specific Smads (Smad1, Smad5, and Smad8), allowing thesereceptor-associated Smads to form a complex with Smad4 and move into thenucleus where the Smad complex binds a DNA binding protein and acts as atranscriptional enhancer. BMPs have a significant role in bone andcartilage formation in vivo. It has been reported that most BMPs areable to stimulate osteogenesis in mature osteoblasts, while BMP-2, 6,and 9 may play an important role in inducing osteoblast differentiationof mesenchymal stem cells. Cheng, H. et al., J. Bone & Joint Surgery 85:1544-52 (2003).

The terms “cancellous bone” or “trabecular bone” as used herein refer tothe spongy bone found in the inner parts of compact bone in which thematrix forms a lattice of large plates and rods known as the trabeculae,which anastomose to form a latticework. This latticework partiallyencloses many intercommunicating spaces filled with bone marrow. Themarrow spaces are relatively large and irregularly arranged, and thebone substance is in the form of slender anastomosing trabeculae andpointed spicules.

The term “chemokine” as used herein refers to a class of chemotacticcytokines that signal leukocytes to move in a specific direction.

The terms “chemotaxis” or “chemotactic” refer to the directed motion ofa motile cell or part along a chemical concentration gradient towardsenvironmental conditions it deems attractive and/or away fromsurroundings it finds repellent.

The term “chondrocytes” as used herein refers to cells found incartilage that produce and maintain the cartilaginous matrix for, forexample, joints, ear canals, trachea, epiglottis, larynx, the discsbetween vertebrae and the ends of ribs. From least to terminallydifferentiated, the chondrocytic lineage is (i) Colony-formingunit-fibroblast (CFU-F); (ii) mesenchymal stem cell/marrow stromal cell(MSC); (iii) chondrocyte.

The term “chondrogenesis” as used herein refers to the formation of newcartilage from cartilage forming or chondrocompetent cells.

The term “chondrogenic” as used herein refers to a potential ofprecursor cells to differentiate into cartilage forming orchondrocompetent cells, wherein the chondrogenic cells include one ormore of a chondrogenic stem cell, a tissuegenic cell, a precursor cell,a progenitor cell, an immature cell, a non-terminally differentiatedcell, a cell with differentiation potential, or a combination thereof.

The terms “cortical bone” or “compact bone” as used herein refer to thedense outer layer of bone that consists largely of concentric lamellarosteons and interstitial lamellae. The spaces or channels are narrow andthe bone substance is densely packed.

The term “interleukin” as used herein refers to a cytokine secreted bywhite blood cells as a means of communication with other white bloodcells.

The term “cytokine” as used herein refers to small soluble proteinsubstances secreted by cells which have a variety of effects on othercells. Cytokines mediate many important physiological functionsincluding growth, development, wound healing, and the immune response.They act by binding to their cell-specific receptors located in the cellmembrane, which allows a distinct signal transduction cascade to startin the cell, which eventually will lead to biochemical and phenotypicchanges in target cells. Generally, cytokines act locally. They includetype I cytokines, which encompass many of the interleukins, as well asseveral hematopoietic growth factors; type II cytokines, including theinterferon's and interleukin-10; tumor necrosis factor (“TNF”)-relatedmolecules, including TNFα and lymphotoxin; immunoglobulin super-familymembers, including interleukin 1 (“IL-1”); and the chemokines, a familyof molecules that play a critical role in a wide variety of immune andinflammatory functions. The same cytokine can have different effects ona cell depending on the state of the cell. Cytokines often regulate theexpression of, and trigger cascades of other cytokines. Nonlimitingexamples of cytokines include e.g., IL-1α, IL-β, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12/IL-23 P40, IL13, IL-17,IL-18, TGF-β, IFN-γ, GM-CSF, Groα, MCP-1 and TNF-α.

The term “decellularize” refers to the removal of at least a portion ofthe endogenous cells from a tissue. The term “devitalize” refers to thekilling of cells which could occur with or without cell removal.

The term “Demineralized bone matrix” (DBM) refers to a bone-derivedmaterial that has osteoconductive and osteoinductive activity. DBM maybe prepared by either acid extraction or non-acid extraction ofallograft bone, resulting in loss of most of the mineralized componentbut retention of collagen and noncollagenous proteins, including growthfactors. Methods for preparing demineralized bone matrix from bone areknown in the art, as disclosed, for example, in U.S. Pat. Nos.5,073,373; 5,484,601; and 5,284,655, which are incorporated herein byreference. DBM may be prepared from autologous bone, allogeneic (or“allograft”) bone, or xenogeneic bone. DBM may be prepared fromcancellous bone, cortical bone, or combinations of cancellous andcortical bone. For the purpose of the present disclosure, demineralizedbone includes bone matrix having a residual mineral content of 8% orless (w/w), 5% or less (w/w), 2% or less (w/w), 1% or less (w/w), 0.5%or less (w/w), or consisting essentially of collagen, non-collagenproteins such as growth factors, and other nonmineral substances foundin the original bone, although not necessarily in their originalquantities. Partially demineralized bone includes bone matrix having anymineral content removed relative to naturally occurring bone, suchmineral content could be 20% or less (w/w), 15% or less (w/w), 10% orless (w/w), or lower. The term “demineralized cortical bone” (DCB) asused herein refers to a demineralized allograft cortical bone

The term “derivative” as used herein means a compound that may beproduced from another compound of similar structure in one or moresteps. A “derivative” or “derivatives” of a peptide or a compoundretains at least a degree of the desired function of the peptide orcompound. Accordingly, an alternate term for “derivative” may be“functional derivative.” Derivatives can include chemical modificationsof the peptide, such as akylation, acylation, carbamylation, iodinationor any modification that derivatizes the peptide. Such derivatizedmolecules include, for example, those molecules in which free aminogroups have been derivatized to form amine hydrochlorides, p-toluenesulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups,chloroacetyl groups or formal groups. Free carboxyl groups can bederivatized to form salts, esters, amides, or hydrazides. Free hydroxylgroups can be derivatized to form O-acyl or O-alkyl derivatives. Theimidazole nitrogen of histidine can be derivatized to formN-im-benzylhistidine. Also included as derivatives or analogues arethose peptides that contain one or more naturally occurring amino acidderivative of the twenty standard amino acids, for example,4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, homoserine,ornithine or carboxyglutamiate, and can include amino acids that are notlinked by peptide bonds. Such peptide derivatives can be incorporatedduring synthesis of a peptide, or a peptide can be modified bywell-known chemical modification methods (see, e.g., Glazer et al.,Chemical Modification of Proteins, Selected Methods and AnalyticalProcedures, Elsevier Biomedical Press, New York (1975)).

The term “differentiation” as used herein refers to the process ofdevelopment with an increase in the level of organization or complexityof a cell or tissue, accompanied with a more specialized function.

The term “nonexpanded” as used herein refers to a cell population thathas not been grown in culture (in vitro) to increase the number of cellsin the cell population.

The term “endogenous” as used herein refers to that which is naturallyoccurring, incorporated within, housed within, adherent to, attached toor resident in.

The term “extracellular matrix” as used herein refers to a scaffold in acell's external environment with which the cell interacts via specificcell surface receptors. The extracellular matrix serves many functions,including, but not limited to, providing support and anchorage forcells, segregating one tissue from another tissue, and regulatingintracellular communication. The extracellular matrix is composed of aninterlocking mesh of fibrous proteins and glycosaminoglycans (GAGs).Examples of fibrous proteins found in the extracellular matrix includecollagen, elastin, fibronectin, and laminin. Examples of GAGs found inthe extracellular matrix include proteoglycans (e.g., heparin sulfate),chondroitin sulfate, keratin sulfate, and non-proteoglycanpolysaccharide (e.g., hyaluronic acid). The term “proteoglycan” refersto a group of glycoproteins that contain a core protein to which isattached one or more glycosaminoglycans.

The term “factors” as used herein refers to nonliving components thathave a chemical or physical effect. For example, a “paracrine factor” isa diffusible signaling molecule that is secreted from one cell type thatacts on another cell type in a tissue. The term “secreted factors” referto factors that are secreted or otherwise produced by cells, such asextracellular macromolecules, growth factors, cytokines and adipokines,into e.g., the surrounding extracellular matrix or medium.

The term “graft” as used herein refers to a tissue, biologic fluid ororgan transplanted from a donor to a recipient. It includes, but is notlimited to, a self-tissue transferred from one body site to another inthe same individual (“autologous graft”), a tissue transferred betweengenetically identical individuals or sufficiently immunologicallycompatible to allow tissue transplant (“syngeneic graft”), a tissuetransferred between genetically different members of the same species(“allogeneic graft” or “allograft”), and a tissue transferred betweendifferent species (“xenograft”). The terms “graft” and “tissue graft”can be used interchangebly.

The term “growth factor” as used herein refers to extracellularpolypeptide molecules that bind to a cell-surface receptor triggering anintracellular signaling pathway, leading to proliferation,differentiation, or other cellular response.

The term “growth induction” as used herein refers to a process by whichcells are stimulated to grow, develop, differentiate, de-differentiateand/or trans-differentiate into a population of at least partiallyidentical cells or an ensemble of cells that are not necessarilyidentical. This ensemble of cells may be a tissue or an organ.

The term “growth-inductive components” refers to biological and/orchemical factors or substances that stimulate cells to grow, develop,differentiate, de-differentiate and/or trans-differentiate into apopulation of at least partially identical cells or an ensemble of cellsthat are not necessarily identical. Growth-inductive components include,but are not limited to, mitogens, growth factors and cytokines.Exemplary growth-inductive components include, but are not limited to,ions (e.g., calcium); hormones; steroids (e.g., estrogens); terpenoids(e.g., retinoic acid); peptides (e.g., Parathyroid hormone (PTH),parathyroid hormone-related peptide (PTHrP), insulin growth factors(e.g., TGF-β, BMPs, IGF-1, VEGF, PDGF, FGF); transcription factors(e.g., Wnt, SOX-9); eicosanoids (e.g., prostaglandins); catabolicinterleukins (e.g., IL-1); and anabolic interleukins (e.g., IL-6, IL-4and IL-10). Other growth-inductive components are listed in Gaissmaieret al. (2008), Int. J. Care Injured, 39S1: S88-S96, the entire contentsof which are incorporated by reference herein.

The terms “marker” or “cell surface marker” are used interchangeablyherein to refer to an antigenic determinant or epitope found on thesurface of a specific type of cell. Cell surface markers can facilitatethe characterization of a cell type, its identification, and eventuallyits isolation. Cell sorting techniques are based on cellular biomarkerswhere a cell surface marker(s) may be used for either positive selectionor negative selection, i.e., for inclusion or exclusion, from a cellpopulation.

The term “matrix” refers to a surrounding substance within whichsomething is contained or embedded.

The term “mesenchymal stem cells (MSCs)” as used herein refers tonon-blood adult stem cells found in a variety of tissues. They arecharacterized by their spindle-shape morphologically; by the expressionof specific markers on their cell surface; and by their ability underappropriate conditions, to differentiates along a minimum of threelineages (osteogenic, chondrogenic and adipogenic). When referring tobone or cartilage, MSCs commonly are known as osteochondrogenic,osteogenic, or chondrogenic, since a single MSC has shown the ability todifferentiate into chondrocytes or osteoblasts, depending on the medium.

MSCs secrete many biologically important molecules, includinginterleukins 6, 7, 8, 11, 12, 14, and 15, M-CSF, Flt-3 ligand, SCF, LIF,bFGF, VEGF, P1GF and MCP1 (Majumdar et al., J. Cell Physiol. 176: 57-66(1998), Kinnaird et al, Circulation 109: 1543-49 (2004)). In 2004, itwas reported that no single marker that definitively identifies MSCs invivo had yet been identified, due to the lack of consensus from diversedocumentations of the MSC phenotype. Baksh et al., J. Cell. Mol. Med.2004, 8(3) 301-16, 305. There is general agreement that MSCs lacktypical hematopoietic antigens, namely CD14, CD34, and CD45. (Id.;citing Pittenger, et al., Science. 1999. 284: 143-47).

The term “multipotent” as used herein refers to a cell capable of givingrise to a limited number of cell types of a particular cell line.

The term “myogenic” refers to a potential of precursor cells todifferentiate into a muscle forming or myocompetent cells, wherein themyogenic cells include one or more of a myogenic stem cell, atissuegenic cell, a precursor cell, a progenitor cell, an immature cell,a non-terminally differentiated cell, a cell with differentiationpotential, or a combination thereof.

The term “osteoblasts” as used herein refers to cells that arise whenosteoprogenitor cells or mesenchymal cells, which are located near allbony surfaces and within the bone marrow, differentiate under theinfluence of growth factors. Osteoblasts, which are responsible for bonematrix synthesis, secrete a collagen rich ground substance essential forlater mineralization of hydroxyapatite and other crystals. The collagenstrands form osteoids (spiral fibers of bone matrix). Osteoblasts causecalcium salts and phosphorus to precipitate from the blood, which bondwith the newly formed osteoid to mineralize the bone tissue. Onceosteoblasts become trapped in the matrix they secrete, they becomeosteocytes. From least to terminally differentiated, the osteocytelineage is (i) Colony-forming unit-fibroblast (CFU-F); (ii) mesenchymalstem cell/marrow stromal cell (MSC); (iii) osteoblast; and (iv)osteocyte.

The term “osteocalcin” as used herein refers to a protein constituent ofbone; circulating levels are used as a marker of increased boneturnover.

The term “osteoclast” as used herein refers to large multinucleate cellsassociated with areas of bone resorption (breakdown).

The term “osteoconduction” as used herein refers to a process by whichbone is directed so as to conform to a material's surface. Anosteoconductive surface is one that permits bone growth on its surfaceor down into pores, channels or pipes. Osteoconductive materialfacilitates the spontaneous formation of bone by furnishing amicroenvironment that supports the ingrowth of blood vessels,perivascular tissue and osteoprogenitor cells into the site where it isdeposited. Examples of osteoconductive materials, include, but notlimited to, processed human bone (allograft bone), purified collagen,calcium phosphate ceramics, synthetic polymers, BMP-2 and 4, VEGF, bFGF,TGF-β, and PDGF.

The term “osteoconductive matrix” as used herein refers to a matrix thatis inert in and of itself but on which cells can climb and grow bone.

The term “osteogenic” refers to a potential of precursor cells todifferentiate into bone forming or osteocompetent cells, wherein theosteogenic cells include one or more of an osteogenic stem cell, atissuegenic cell, a precursor cell, a progenitor cell, an immature cell,a non-terminally differentiated cell, a cell with differentiationpotential, or a combination thereof.

The term “osteogenesis” as used herein refers to the development orformation of new bone by bone forming or osteocompetent cells.

The term “osteoinduction” as used herein refers to a process by whichprimitive, undifferentiated or non-terminally differentiated pluripotentcells are stimulated to develop into a bone forming cell lineage therebyinducing osteogenesis. For example, the majority of bone healing in afracture is dependent on osteoinduction. Osteoinductive materials can begenerated by combining a porous scaffold with osteogenic cells and/orosteoinductive components, including, but not limited to, growth factorssuch as BMP-2 and 4, VEGF, bFGF, TGF-β, and PDGF.

The term “osteoinductive matrix” as used herein refers to a matrixcontaining a substance or substances that recruit local cells to induce(meaning to cause, bring about, bring about, or trigger) local cells toproduce bone.

The terms “osteoinductive components” or “osteogenic factors” are usedinterchangeably to refer to the plethora of mediators associated withbone development and repair, including, but not limited to, bonemorphogenic proteins (BMPs), vascular endothelial growth factor (VEGF),basic fibroblast growth factor (bFGF), transforming growth factor beta(TGFβ), and platelet-derived growth factor (PDGF).

The term “osteointegration” refers to an anchorage mechanism wherebynonvital components can be incorporated reliably into living bone andthat persist under all normal conditions of loading.

The term “periosteum” as used herein refers to the normal investment ofbone, consisting of a dense, fibrous outer layer, to which musclesattach, and a more delicate, inner layer capable of forming bone.

The term “Platelet Derived Growth Factor” (PDGF) as used herein refersto a major mitogen for connective tissue cells and certain other celltypes. It is a dimeric molecule consisting of disulfide-bonded,structurally similar A and B-polypeptide chains, which combine to homo-and hetero-dimers. The PDGF isoforms exert their cellular effects bybinding to and activating two structurally related protein tyrosinekinase receptors, the α-receptor and the β-receptor. Activation of PDGFreceptors leads to stimulation of cell growth, but also to changes incell shape and motility; PDGF induces reorganization of the actinfilament system and stimulates chemotaxis, i.e., a directed cellmovement toward a gradient of PDGF. In vivo, PDGF plays a role inembryonic development and during wound healing.

The term “pluripotent” as used herein refers to the ability to developinto multiple cells types, including all three embryonic lineages,forming the body organs, nervous system, skin, muscle and skeleton.

The terms “priming” “pre-conditioning” and “conditioning” are usedinterchangeably herein and refer to the use of one or more stimuli toprepare a tissue for use in grafting. The term “non-primed” tissuerefers to a tissue that is otherwise treated in the same manner exceptthe priming condition.

The term “progenitor cell” as used herein refers to an early descendantof a stem cell that can only differentiate, but can no longer renewitself. Progenitor cells mature into precursor cells that mature intomature phenotypes. Hematopoietic progenitor cells are referred to ascolony-forming units (CFU) or colony-forming cells (CFC). The specificlineage of a progenitor cell is indicated by a suffix, such as, but notlimited to, CFU-E (erythrocytic), CFU-F (fibroblastic), CFU-GM(granulocytic/macrophage), and CFU-GEMM (pluripotent hematopoieticprogenitor). Osteoclasts arise from hematopoietic cells of themonocyte/neutrophil lineage (CFU-GM). Osteoprogenitor cells arise frommesenchymal stem cells and are committed to an osteocyte lineage.

The term “regeneration” or “regenerate” as used herein refers to aprocess of recreation, reconstitution, renewal, revival, restoration,differentiation and growth to form a tissue with characteristics thatconform with a natural counterpart of the tissue.

The term “resident,” and its various grammatical forms, as used hereinrefers to being present habitually, existing in or intrinsic to orincorporated therein.

The term “scaffold” as used herein refers to a structure capable ofsupporting a three-dimensional tissue formation. A three-dimensionalscaffold is believed to be critical to replicate the in vivo milieu andto allow the cells to influence their own microenvironment. Scaffoldsmay serve to promote cell attachment and migration, to deliver andretain cells and biochemical factors, to enable diffusion of vital cellnutrients and expressed products, and to exert certain mechanical andbiological influences to modify the behavior of the cell phase. Ascaffold utilized for tissue reconstruction has several requisites. Sucha scaffold should have a high porosity and an adequate pore size tofacilitate cell seeding and diffusion of both cells and nutrientsthroughout the whole structure. Biodegradability of the scaffold is alsoan essential requisite. The scaffold should be absorbed by thesurrounding tissues without the necessity of a surgical removal, suchthat the rate at which degradation occurs coincides as closely aspossible with the rate of tissue formation. As cells are fabricatingtheir own natural matrix structure around themselves, the scaffoldprovides structural integrity within the body and eventually degradesleaving the neotissue (newly formed tissue) to assume the mechanicalload.

The term “stem cells” refers to undifferentiated cells having highproliferative potential with the ability to self-renew (make more stemcells by cell division) that can generate daughter cells that canundergo terminal differentiation into more than one distinct cellphenotype.

The term “stimuli” or “stimulating agent” as used herein refers to anenvironmental (e.g., microenvironmental) or external force, condition,factor or substance that exerts some change or effect, in particular inthe context of priming a tissue for grafting.

The phrase “recipient” or “subject in need thereof” as used hereinrefers to a patient that (i) will receive or be administered at leastone graft (e.g., allograft), (ii) is receiving or administered at leastone graft (e.g., allograft); or (iii) has received or administered atleast one graft (e.g., allograft), unless the context and usage of thephrase indicates otherwise. The term “donor” refers to a patient whoprovides at least one graft. Donor and recipient can be the same ordifferent.

The term “tissuegenic” as used herein refers to a potential of anprecursor cell to differentiate into a mature cell type and toregenerate a tissue, wherein the tissuegenic cells include one or moreof a tissuegenic stem cell, a tissuegenic cell, a precursor cell, aprogenitor cell, an immature cell, a non-terminally differentiated cell,a cell with differentiation potential, or a combination thereof.Exemplary tissuegenic cells include but are not limited to a stem cell,a progenitor cell or a combination thereof. The term “osteogenic” refersmore specifically to cell differentiation and tissue regeneration withregard to bone.

The term “vascularization” as used herein refers to a process ofingrowth of blood vessels and perivascular tissue within agrowth-conductive matrix to support the deposition and adhesion oftissuegenic cells to effect tissue regeneration.

The terms “VEGF”, “VEGF-1” or “vascular endothelial growth factor-1” areused interchangeably herein to refer to a cytokine that mediatesnumerous functions of endothelial cells including proliferation,migration, invasion, survival, and permeability. The term “VEGF-2”refers to a regulator for growth of vascular endothelial and smoothmuscle cells. VEGF-2 stimulates the growth of human vascular endothelialcells but inhibits growth of human aortic smooth muscle cells induced byplatelet-derived growth factor.

The term “viable” as used herein refers to having the ability to grow,expand, or develop; capable of living.

The term “xenogeneic” as used herein refers to cells or tissues derivedfrom individuals of different species, including, but not limited to,porcine, bovine, caprine, equine, canine, lapine, feline, and/ornon-human mammals, such as, but not limited to, whale, and porpoise.

2. Tissue Compartments and Cells Therein

The methods and compositions of the present invention can be applied toany tissue or organ suitable for grafting. Some exemplary tissues andassociated cells are described herein. One or ordinary skill in the artwould understand that the present invention is applicable to othertissue and organ types as well.

In multicellular organisms, cells that are specialized to perform commonfunctions are usually organized into cooperative assemblies embedded ina complex network of secreted extracellular macromolecules, theextracellular matrix (ECM), to form specialized tissue compartments.Individual cells in such tissue compartments are in contact with ECMmacromolecules. The ECM helps hold the cells and compartments togetherand provides an organized lattice or scaffold within which cells canmigrate and interact with one another. In many cases, cells in acompartment can be held in place by direct cell-cell adhesions. Invertebrates, such compartments may be of four major types, a connectivetissue (CT) compartment, an epithelial tissue (ET) compartment, a muscletissue (MT) compartment and a nervous tissue (NT) compartment, which arederived from three embryonic germ layers: ectoderm, mesoderm andendoderm. The NT and portions of the ET compartments are differentiatedfrom the ectoderm; the CT, MT and certain portions of the ETcompartments are derived from the mesoderm; and further portions of theET compartment are derived from the endoderm.

The connective tissue compartment contains cells that primarily functionto elaborate and maintain ECM structure. The character of theextracellular matrix is region-specific and is determined by the amountof the extracellular materials.

Common cell types of connective tissue compartments include:fibroblasts, macrophages, mast cells, and plasma cells. Specializedconnective tissue compartments, such as cartilage, bone, and thevasculature, and those with special properties, such as adipose,tendons, ligaments, etc., have specialized cells to perform specializedfunctions.

2.1. Adipose

2.1.1. Adipose Tissue Compartment

Adipose tissue compartments are dynamic, multifunctional, ubiquitous andloose connective tissue compartments. Adipose comprises fibroblasts,smooth muscle cells, endothelial cells, leukocytes, macrophages, andclosely packed mature lipid-filled fat cells, termed adipocytes, withcharacteristic nuclei pushed to one side, embedded within an areolarmatrix that are located in subcutaneous layers of skin and muscle(panniculus adiposus), in the kidney region, cornea, breasts,mesenteries, mediastinium, and in the cervical, axillary and inguinalregions. Adipocytes play a primary role in energy storage and inproviding insulation and protection. As sites of energy storage,adipocytes regulate the accumulation or mobilization of triacylglycerolin response to the body's energy requirements and store energy in theform of a single fat droplet of triglycerides.

Each adipocyte is surrounded by a thick ECM called the basal lamina. Thestrong adipocyte ECM scaffold lowers mechanical stress by spreadingforces over a large surface area of the adipose tissue compartments. TheECM composition of adipocytes is similar to that of other cell types,but it is the relative quantity of individual components that impartcell specificity. Adipocyte ECM is particularly enriched in collagen VI,a coiled coil comprising α1(VI), α2(VI) and α3(VI) subunits. Collagen VIbinds to collagen IV and also to other matrix proteins such asproteoglycans and fibronectin. The core proteins associated with theadipocyte ECM, which have been identified through with current proteomictechniques, have been reviewed by Mariman et al. (Cell. Mol. Life Sci.,2010, 67:1277-1292).

Adipocyte ECM undergoes biphasic development during adipogenesis, theprocess of formation of mature adipose tissue compartments. There is aninitial decrease in collagen I and III, whereas their levels come backto pre-differentiation state at later stages. Mature adipocyte ECM ismaintained in a dynamic state with constant turnover of ECM componentsby a balance of activities of ECM constructive enzymes and ECMdegradation enzymes. In early stages of differentiation, the balance isshifted towards the constructive factors. (Mariman et al., 2010, Cell.Mol. Life Sci., 67:1277-1292). Maturation of newly synthesized ECMcomponents is initiated in the ER lumen where ECM proteins undergobiochemical modifications and proteolytic processing prior to assembly.For collagen, such modifications include proline- andlysine-hydroxylation and glycosylation and clipping of N- and C-terminalpeptides by respective procollagen-N- and -C-collagenase. Processedproteins are then assembled and secreted into the extracellularenvironment where they undergo further processing by secretedextracellular modification and processing enzymes. As the preadipocytesdifferentiate and begin to store fat, ECM assumes a basal laminarstructure.

2.1.2. Adipose-Derived Stem Cells

Adipose also comprises a population of pluripotent stem cells that havethe potential to give rise to cells of all three embryonic lineages:ectodermal, mesodermal and endodermal. Adipogenesis, which comprises thesteps of differentiation of such pluripotent cells to mature adipocytes,is initiated by differentiation of these pluripotent cells to give riseto a population of mesenchymal precursor cells or mesenchymal stem cells(MSCs), which have the potential to differentiate into a variety ofmesodermal cell lineages such as for example, myoblasts, chondroblasts,osteoblasts and adipocytes. In the presence of appropriate environmentaland gene expression signals, the MSCs go through growth arrest anddifferentiate into precursors with a determined fate that undergo clonalexpansion, become committed and terminally differentiate to give rise tomature cells. The population of MSCs and more committed adiposeprogenitors that are found along with the stroma of adipose tissuecollectively are termed adipose-derived stem cells (ASCs). These cellshave a characteristic CD45−CD31−CD34+CD105+ surface phenotype. In thecase of adipocyte differentiation, ASCs differentiate to proadipocytesthat undergo final differentiation to give rise to mature adipocytes.Mesenchymal progenitor cells with chondrogenic potential have also beenidentified in the infrapatellar fat pad in joints. (Lee et al., TissueEngg. 2010, 16(1): 317-325). Cell lineages and respective inductivefactors that can be derived from ASC lines have been reviewed by Brownet. al., Plast. Reconstr. Surg., 2010, 126(6): 1936-1946; Gregoire etal., Physiol. Rev., 1998, 78(3): 783-809).

2.1.3. Adipose Secreted Factors

Adipose is considered a secretory organ. The adipose secretome not onlyincludes structural and soluble factors contributing to the formation ofthe adipose matrix, but also a horde of soluble factors with endocrinefunction, such as growth factors, hormones, chemokines and lipids,collectively termed adipokines. Exemplary adipokines include, withoutlimitation, leptin, adiponectin, resistin, interleukin 6 (IL-6),monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor alpha(TNF-α); fibroblast growth factor (FGF), and vascular endothelial growthfactor (VEGF). Exemplary immunogical adipokines, particularly involvedin inflammatory pathways include, without limitation, serum amyloid A3(SAA3), IL-6, adiponectin, TNF-α and haptoglobin. Exemplary adipokinesinvolved in the production of new blood vessels include, withoutlimitation, angiopoietin-1, angiopoietin-2, VEGF, transforming growthfactor beta (TGF-β), hepatic growth factor (HGF), stromal derived growthfactor 1 (SDF-1), TNF-α, resistin, leptin, tissue factor, placentalgrowth factor (PGF), insulin like growth factor (IGF), and monobutyrin.

Adiponectin, a key metabolic factor secreted from adipocytes, is a30-KDa protein that may exist as a trimer, low molecular weight hexamersor high molecular weight 18mers. Adiponectin circulates throughout theplasma and has a variety of metabolic effects including, but not limitedto, glucose lowering and cardioprotection stimulation of smooth muscleproliferation. Adiponectin has been implicated in a number ofpathological conditions including, but not limited to diabetes, obesity,metabolic syndrome, cardiovascular disease and wound healing.

Resistin, a member of the resistin-like (RELM) hormone family, issecreted by stromal vascular cells of adipose. Resistin is secreted intwo multimeric isoforms and functions to counterbalance the insulinsensitizing effects of adiponectin. (Truillo, M. E. and Scherer P. E.,Endocrine Rev. 2006, 27(7): 762-778).

Secretions from resident adipocytes, macrophages and ASCs collectivelycontribute to the adipose secretome. Angiogenic, hematopoietic, andproinflammatory adipokine profiles of ASCs are reported by Kilroy et.al., Cell. Physiol., 2007, J. 212: 702-709.)

2.2. Bone

2.2.1. Osseous Tissue Compartment

Osseous tissue is a rigid form of connective tissue normally organizedinto definite structures, the bones. These form the skeleton, serve forthe attachment and protection of the soft parts, and, by theirattachment to the muscles, act as levers that bring about body motion.Bone is also a storage place for calcium that can be withdrawn whenneeded to maintain a normal level of calcium in the blood.

Bones can be classified according to their shape. Examples of bone typesinclude: long bones whose length is greater than their widths (e.g.,femur (thigh bone), humerus (long bone of the upper limb), tibia (shinbone), fibula (calf bone), radius (the outer of the two bones of theforearm), and ulna (inner of two bones of the forearm)), short boneswhose length and width is approximately equal (e.g., carpals bones(wrist bones in the hand)), flat bones (e.g., cranium (skull bonessurrounding the brain), scapula (shoulder blade), and ilia (theuppermost and largest bone of the pelvis)), irregular bones (e.g.,vertebra), and Sesamoid bones, small bones present in the joints toprotect tendons (fibrous connective tissues that connect muscles to thebones, e.g., patella bones (knee cap). Grossly, two types of bone may bedistinguished: cancellous, trabecular or spongy bone, and cortical,compact, or dense bone.

Cortical bone, also referred to as compact bone or dense bone, is thetissue of the hard outer layer of bones, so-called due to its minimalgaps and spaces. This tissue gives bones their smooth, white, and solidappearance. Cortical bone consists of haversian sites (the canalsthrough which blood vessels and connective tissue pass in bone) andosteons (the basic units of structure of cortical bone comprising ahaversian canal and its concentrically arranged lamellae), so that incortical bone, bone surrounds the blood supply. Cortical bone has aporosity of about 5% to about 30%, and accounts for about 80% of thetotal bone mass of an adult skeleton.

2.2.2. Cancellous Bone (Trabecular or Spongy Bone)

Cancellous bone tissue, an open, cell-porous network also calledtrabecular or spongy bone, fills the interior of bone and is composed ofa network of rod- and plate-like elements that make the overallstructure lighter and allows room for blood vessels and marrow so thatthe blood supply surrounds bone. Cancellous bone accounts for theremaining 20% of total bone mass but has nearly ten times the surfacearea of cortical bone. It does not contain haversian sites and osteonsand has a porosity of about 30% to about 90%.

The head of a bone, termed the epiphysis, has a spongy appearance andconsists of slender irregular bone trabeculae, or bars, which anastomoseto form a lattice work, the interstices of which contain the marrow,while the thin outer shell appears dense. The irregular marrow spaces ofthe epiphysis become continuous with the central medullary cavity of thebone shaft, termed the diaphysis, whose wall is formed by a thin plateof cortical bone.

Both cancellous and cortical bone have the same types of cells andintercellular substance, but they differ from each other in thearrangement of their components and in the ratio of marrow space to bonesubstance. In cancellous bone, the marrow spaces are relatively largeand irregularly arranged, and the bone substance is in the form ofslender anastomosing trabeculae and pointed spicules. In cortical bone,the spaces or channels are narrow and the bone substance is denselypacked.

With very few exceptions, the cortical and cancellous forms are bothpresent in every bone, but the amount and distribution of each type varyconsiderably. The diaphyses of the long bones consist mainly of corticaltissue; only the innermost layer immediately surrounding the medullarycavity is cancellous bone. The tabular bones of the head are composed oftwo plates of cortical bone enclosing marrow space bridged by irregularbars of cancellous bone. The epiphyses of the long bones and most of theshort bones consist of cancellous bone covered by a thin outer shell ofcortical bone.

Each bone, except at its articular end, is surrounded by a vascularfibroelastic coat, the periosteum. The so-called endosteum, or innerperiosteum of the marrow cavity and marrow spaces, is not awell-demarcated layer; it consists of a variable concentration ofmedullary reticular connective tissue that contains osteogenic cellsthat are in immediate contact with the bone tissue.

2.2.3. Components of Bone

Bone is composed of cells and an intercellular matrix of organic andinorganic substances. The organic fraction consists of collagen,glycosaminoglycans, proteoglycans, and glycoproteins. The protein matrixof bone largely is composed of collagen, a family of fibrous proteinsthat have the ability to form insoluble and rigid fibers. The maincollagen in bone is type I collagen. The inorganic component of bone,which is responsible for its rigidity and may constitute up totwo-thirds of its fat-free dry weight, is composed chiefly of calciumphosphate and calcium carbonate, in the form of calcium hydroxyapatite,with small amounts of magnesium hydroxide, fluoride, and sulfate. Thecomposition varies with age and with a number of dietary factors. Thebone minerals form long fine crystals that add strength and rigidity tothe collagen fibers; the process by which it is laid down is termedmineralization.

2.2.4. Bone Cells and Secreted Factors

Four cell types in bone are involved in its formation and maintenance.These are 1) osteoprogenitor cells, 2) osteoblasts, 3) osteocytes, and4) osteoclasts.

Osteoprogenitor cells arise from mesenchymal cells, and occur in theinner portion of the periosteum and in the endosteum of mature bone.They are found in regions of the embryonic mesenchymal compartment wherebone formation is beginning and in areas near the surfaces of growingbones. Structurally, osteoprogenitor cells differ from the mesenchymalcells from which they have arisen. They are irregularly shaped andelongated cells having pale-staining cytoplasm and pale-staining nuclei.Osteoprogenitor cells, which multiply by mitosis, are identified chieflyby their location and by their association with osteoblasts. Someosteoprogenitor cells differentiate into osteocytes. While osteoblastsand osteocytes are no longer mitotic, it has been shown that apopulation of osteoprogenitor cells persists throughout life.

Osteoblasts, which are located on the surface of osteoid seams (thenarrow region on the surface of a bone of newly formed organic matrixnot yet mineralized), are derived from osteoprogenitor cells. They areimmature, mononucleate, bone-forming cells that synthesize collagen andcontrol mineralization. Osteoblasts can be distinguished fromosteoprogenitor cells morphologically; generally they are larger thanosteoprogenitor cells, and have a more rounded nucleus, a more prominentnucleolus, and cytoplasm that is much more basophilic. Osteoblasts makea protein mixture known as osteoid, primarily composed of type Icollagen, which mineralizes to become bone. Osteoblasts also manufacturehormones, such as prostaglandins, alkaline phosphatase, an enzyme thathas a role in the mineralization of bone, and matrix proteins.

Osteocytes, star-shaped mature bone cells derived from ostoblasts andthe most abundant cell found in compact bone, maintain the structure ofbone. Osteocytes, like osteoblasts, are not capable of mitotic division.They are actively involved in the routine turnover of bony matrix andreside in small spaces, cavities, gaps or depressions in the bone matrixcalled lacuna. Osteocytes maintain the bone matrix, regulate calciumhomeostasis, and are thought to be part of the cellular feedbackmechanism that directs bone to form in places where it is most needed.Bone adapts to applied forces by growing stronger in order to withstandthem; osteocytes may detect mechanical deformation and mediatebone-formation by osteoblasts.

Osteoclasts, which are derived from a monocyte stem cell lineage andpossess phagocytic-like mechanisms similar to macrophages, often arefound in depressions in the bone referred to as Howship's lacunae. Theyare large multinucleated cells specialized in bone resorption. Duringresorption, osteoclasts seal off an area of bone surface; then, whenactivated, they pump out hydrogen ions to produce a very acidenvironment, which dissolves the hydroxyapatite component. The numberand activity of osteoclasts increase when calcium resorption isstimulated by injection of parathyroid hormone (PTH), while osteoclasticactivity is suppressed by injection of calcitonin, a hormone produced bythyroid parafollicular cells.

The bone matrix accounts for about 90% of the total weight of compactbone and is composed of microcrystalline calcium phosphate resemblinghydroxyapatite (60%) and fibrillar type I collagen (27%). The remaining3% consists of minor collagen types and other proteins includingosteocalcin, osteonectin, osteopontin, bone sialoprotein, as well asproteoglycans, glycosaminoglycans, and lipids.

Bone matrix is also a major source of biological information thatskeletal cells can receive and act upon. For example, extracellularmatrix glycoproteins and proteoglycans in bone bind a variety of growthfactors and cytokines, and serve as a repository of stored signals thatact on osteoblasts and osteoclasts. Examples of growth factors andcytokines found in bone matrix include, but are not limited to, BoneMorphogenic Proteins (BMPs), Epidermal Growth Factors (EGFs), FibroblastGrowth Factors (FGFs), Platelet-Derived Growth Factors (PDGFs),Insulin-like Growth Factor-1 (IGF-1), Transforming Growth Factors(TGFs), Bone-Derived Growth Factors (BDGFs), Cartilage-Derived GrowthFactor (CDGF), Skeletal Growth Factor (hSGF), Interleukin-1 (IL-1), andmacrophage-derived factors.

There is an emerging understanding that extracellular matrix moleculesthemselves can serve regulatory roles, providing both direct biologicaleffects on cells as well as key spatial and contextual information.

Examples of factors secreted by bone cells include, but are not limitedto, Bone Morphogenic Proteins (BMPs), Epidermal Growth Factors (EGFs),Fibroblast Growth Factors (FGFs), Platelet-Derived Growth Factors(PDGFs), Insulin-like Growth Factor-1 (IGF-1), Transforming GrowthFactors (TGFs), Bone-Derived Growth Factors (BDGFs), Cartilage-DerivedGrowth Factor (CDGF), Skeletal Growth Factor (hSGF), Interleukin-1(IL-1), and macrophage-derived factors.

2.2.5. Periosteum and Endosteum

The periosteum is a fibrous connective tissue investment of bone, exceptat the bone's articular surface. Its adherence to the bone varies bylocation and age. In young bone, the periosteum is stripped off easily.In adult bone, it is more firmly adherent, especially so at theinsertion of tendons and ligaments, where more periosteal fiberspenetrate into the bone as the perforating fibers of Sharpey (bundles ofcollagenous fibers that pass into the outer circumferential lamellae ofbone). The periosteum consists of two layers, the outer of which iscomposed of course, fibrous connective tissue containing few cells butnumerous blood vessels and nerves. The inner layer, which is lessvascular but more cellular, contains many elastic fibers. During growth,an osteogenic layer of primitive connective tissue forms the inner layerof the periosteum. In the adult, this is represented only by a row ofscattered, flattened cells closely applied to the bone. The periosteumserves as a supporting bed for the blood vessels and nerves going to thebone and for the anchorage of tendons and ligaments. The osteogeniclayer, which is considered a part of the periosteum, is known to furnishosteoblasts for growth and repair, and acts as an important limitinglayer controlling and restricting the extend of bone formation. Becauseboth the periosteum and its contained bone are regions of the connectivetissue compartment, they are not separated from each other or from otherconnective tissues by basal laminar material or basement membranes.Perosteal stem cells have been shown to be important in boneregeneration and repair. (Zhang et al., J. Musculoskelet. Neuronal.Interact. 2005. 5(4): 360-362).

The endosteum lines the surface of cavities within a bone (marrow cavityand central canals) and also the surface of trabeculae in the marrowcavity. In growing bone, it consists of a delicate striatum ofmyelogenous reticular connective tissue, beneath which is a layer ofosteoblasts. In the adult, the osteogenic cells become flattened and areindistinguishable as a separate layer. They are capable of transforminginto osteogenic cells when there is a stimulus to bone formation, asafter a fracture.

2.2.6. Bone Marrow

The marrow is a soft connective tissue that occupies the medullarycavity of the long bones, the larger central canals, and all of thespaces between the trabeculae of spongy bone. It consists of a delicatereticular connective tissue, in the meshes of which lie various kinds ofcells. Two varieties of marrow are recognized: red and yellow. Redmarrow is the only type found in fetal and young bones, but in the adultit is restricted to the vertebrae, sternum, ribs, cranial bones, andepiphyses of long bones. It is the chief site for the genesis of bloodcells in the adult body. Yellow marrow consists primarily of fat cellsthat gradually have replaced the other marrow elements. Under certainconditions, the yellow marrow of old or emaciated persons loses most ofits fat and assumes a reddish color and gelatinous consistency, known asgelatinous marrow. With adequate stimulus, yellow marrow may resume thecharacter of red marrow and play an active part in the process of blooddevelopment.

2.2.7. Osteogenesis or Ossification

Osteogenesis or ossification is a process by which the bones are formed.There are three distinct lineages that generate the skeleton. Thesomites generate the axial skeleton, the lateral plate mesodermgenerates the limb skeleton, and the cranial neural crest gives rise tothe branchial arch, craniofacial bones, and cartilage. There are twomajor modes of bone formation, or osteogenesis, and both involve thetransformation of a preexisting mesenchymal tissue into bone tissue. Thedirect conversion of mesenchymal tissue into bone is calledintramembranous ossification. This process occurs primarily in the bonesof the skull. In other cases, mesenchymal cells differentiate intocartilage, which is later replaced by bone. The process by which acartilage intermediate is formed and replaced by bone cells is calledendochondral ossification.

2.2.7.1. Intramembranous Ossification

Intramembraneous ossification is the characteristic way in which theflat bones of the scapula, the skull and the turtle shell are formed. Inintramembraneous ossification, bones develop sheets of fibrousconnective tissue. During intramembranous ossification in the skull,neural crest-derived mesenchymal cells proliferate and condense intocompact nodules. Some of these cells develop into capillaries; otherschange their shape to become osteoblasts, committed bone precursorcells. The osteoblasts secrete a collagen-proteoglycan matrix that isable to bind calcium salts. Through this binding, the prebone (osteoid)matrix becomes calcified. In most cases, osteoblasts are separated fromthe region of calcification by a layer of the osteoid matrix theysecrete. Occasionally, osteoblasts become trapped in the calcifiedmatrix and become osteocytes. As calcification proceeds, bony spiculesradiate out from the region where ossification began, the entire regionof calcified spicules becomes surrounded by compact mesenchymal cellsthat form the periosteum, and the cells on the inner surface of theperiosteum also become osteoblasts and deposit osteoid matrix parallelto that of the existing spicules. In this manner, many layers of boneare formed.

Intramembraneous ossification is characterized by invasion ofcapillaries into the mesenchymal zone, and the emergence anddifferentiation of mesenchymal cells into mature osteoblasts, whichconstitutively deposit bone matrix leading to the formation of bonespicules, which grow and develop, eventually fusing with other spiculesto form trabeculae. As the trabeculae increase in size and number theybecome interconnected forming woven bone (a disorganized weak structurewith a high proportion of osteocytes), which eventually is replaced bymore organized, stronger, lamellar bone.

The molecular mechanism of intramembranoaus ossification involves bonemorphogenetic proteins (BMPs) and the activation of a transcriptionfactor called CBFA1. Bone morphogenetic proteins, for example, BMP2,BMP4, and BMP7, from the head epidermis are thought to instruct theneural crest-derived mesenchymal cells to become bone cells directly.BMPs activate the Cbfal gene in mesenchymal cells. The CBFA1transcription factor is known to transform mesenchymal cells intoosteoblasts. Studies have shown that the mRNA for mouse CBFA1 is largelyrestricted to the mesenchymal condensations that form bone, and islimited to the osteoblast lineage. CBFA1 is known to activate the genesfor osteocalcin, osteopontin, and other bone-specific extracellularmatrix proteins.

2.2.7.2. Endochondral Ossification (Intracartilaginous Ossification)

Endochondral ossification, which involves the in vivo formation ofcartilage tissue from aggregated mesenchymal cells, and the subsequentreplacement of cartilage tissue by bone, can be divided into fivestages. The skeletal components of the vertebral column, the pelvis, andthe limbs are first formed of cartilage and later become bone.

First, the mesenchymal cells are committed to become cartilage cells.This commitment is caused by paracrine factors that induce the nearbymesodermal cells to express two transcription factors, Pax1 andScleraxis. These transcription factors are known to activatecartilage-specific genes. For example, Scleraxis is expressed in themesenchyme from the sclerotome, in the facial mesenchyme that formscartilaginous precursors to bone, and in the limb mesenchyme.

During the second phase of endochondral ossification, the committedmesenchyme cells condense into compact nodules and differentiate intochondrocytes (cartilage cells that produce and maintain thecartilaginous matrix, which consists mainly of collagen andproteoglycans). Studies have shown that N-cadherin is important in theinitiation of these condensations, and N-CAM is important formaintaining them. In humans, the SOX9 gene, which encodes a DNA-bindingprotein, is expressed in the precartilaginous condensations.

During the third phase of endochondral ossification, the chondrocytesproliferate rapidly to form the model for bone. As they divide, thechondrocytes secrete a cartilage-specific extracellular matrix.

In the fourth phase, the chondrocytes stop dividing and increase theirvolume dramatically, becoming hypertrophic chondrocytes. These largechondrocytes alter the matrix they produce (by adding collagen X andmore fibronectin) to enable it to become mineralized by calciumcarbonate.

The fifth phase involves the invasion of the cartilage model by bloodvessels. The hypertrophic chondrocytes die by apoptosis, and this spacebecomes bone marrow. As the cartilage cells die, a group of cells thathave surrounded the cartilage model differentiate into osteoblasts,which begin forming bone matrix on the partially degraded cartilage.Eventually, all the cartilage is replaced by bone. Thus, the cartilagetissue serves as a model for the bone that follows.

The replacement of chondrocytes by bone cells is dependent on themineralization of the extracellular matrix. A number of events lead tothe hypertrophy and mineralization of the chondrocytes, including aninitial switch from aerobic to anaerobic respiration, which alters theircell metabolism and mitochondrial energy potential. Hypertrophicchondrocytes secrete numerous small membrane-bound vesicles into theextracellular matrix. These vesicles contain enzymes that are active inthe generation of calcium and phosphate ions and initiate themineralization process within the cartilaginous matrix. The hypertrophicchondrocytes, their metabolism and mitochondrial membranes altered, thendie by apoptosis.

In the long bones of many mammals (including humans), endochondralossification spreads outward in both directions from the center of thebone. As the ossification front nears the ends of the cartilage model,the chondrocytes near the ossification front proliferate prior toundergoing hypertrophy, pushing out the cartilaginous ends of the bone.The cartilaginous areas at the ends of the long bones are calledepiphyseal growth plates. These plates contain three regions: a regionof chondrocyte proliferation, a region of mature chondrocytes, and aregion of hypertrophic chondrocytes. As the inner cartilagehypertrophies and the ossification front extends farther outward, theremaining cartilage in the epiphyseal growth plate proliferates. As longas the epiphyseal growth plates are able to produce chondrocytes, thebone continues to grow.

2.2.8. Bone Remodeling

Bone constantly is broken down by osteoclasts and re-formed byosteoblasts in the adult. It has been reported that as much as 18% ofbone is recycled each year through the process of renewal, known as boneremodeling, which maintains bone's rigidity. The balance in this dynamicprocess shifts as people grow older: in youth, it favors the formationof bone, but in old age, it favors resorption.

As new bone material is added peripherally from the internal surface ofthe periosteum, there is a hollowing out of the internal region to formthe bone marrow cavity. This destruction of bone tissue is due toosteoclasts that enter the bone through the blood vessels. Osteoclastsdissolve both the inorganic and the protein portions of the bone matrix.Each osteoclast extends numerous cellular processes into the matrix andpumps out hydrogen ions onto the surrounding material, therebyacidifying and solubilizing it. The blood vessels also import theblood-forming cells that will reside in the marrow for the duration ofthe organism's life.

The number and activity of osteoclasts must be tightly regulated. Ifthere are too many active osteoclasts, too much bone will be dissolved,and osteoporosis will result. Conversely, if not enough osteoclasts areproduced, the bones are not hollowed out for the marrow, andosteopetrosis (known as stone bone disease, a disorder whereby the bonesharden and become denser) will result.

2.2.9. Bone Regeneration and Fracture Repair

A fracture, like any traumatic injury, causes hemorrhage and tissuedestruction. The first reparative changes thus are characteristic ofthose occurring in any injury of soft tissue. Proliferating fibroblastsand capillary sprouts grow into the blood clot and injured area, thusforming granulation tissue. The area also is invaded by polymorphonuclear leukocytes and later by macrophages that phagocytize thetissue debris. The granulation tissue gradually becomes denser, and inparts of it, cartilage is formed. This newly formed connective tissueand cartilage is designated as a callus. It serves temporarily instabilizing and binding together the fracture bone. As this process istaking place, the dormant osteogenic cells of the periosteum enlarge andbecome active osteoblasts. On the outside of the fractured bone, atfirst at some distance from the fracture, osseous tissue is deposited.This formation of new bone continues toward the fractured ends of thebone and finally forms a sheath-like layer of bone over thefibrocartilaginous callus. As the amount of bone increases, osteogenicbuds invade the fibrous and cartilaginous callus and replace it with abony one. The cartilage undergoes calcification and absorption in thereplacement of the fibrocartilaginous callus and intramembraneous boneformation also takes place. The newly formed bone is at first a spongyand not a compact type, and the callus becomes reduced in diameter. Atthe time when this subperiosteal bone formation is taking place, bonealso forms in the marrow cavity. The medullary bone growingcentripetally from each side of the fracture unites, thus aiding thebony union.

The process of repair is, in general, an orderly process, but it variesgreatly with the displacement of the fractured ends of the bone and thedegree of trauma inflicted. Uneven or protruding surfaces gradually areremoved, and the healed bone, especially, in young individuals, assumesits original contour.

2.2.10. Osteogenesis and Angiogenesis

Skeletal development and fracture repair includes the coordination ofmultiple events such as migration, differentiation, and activation ofmultiple cell types and tissues. The development of a microvasculatureand microcirculation is important for the homeostasis and regenerationof living bone, without which the tissue would degenerate and die.Recent developments using in vitro and in vivo models of osteogenesisand fracture repair have provided a better understanding of therecruitment nature of the vasculature in skeletal development andrepair.

The vasculature transports oxygen, nutrients, soluble factors andnumerous cell types to all tissues in the body. The growth anddevelopment of a mature vascular structure is one of the earliest eventsin organogenesis. In mammalian embryonic development, the nascentvascular networks develop by aggregation of de novo forming angioblastsinto a primitive vascular plexus (vasculogenesis). This undergoes acomplex remodeling process in which sprouting, bridging and growth fromexisting vessels (angiogenesis) leads to the onset of a functionalcirculatory system.

The factors and events that lead to the normal development of theembryonic vasculature are recapitulated during situations ofneoangiogenesis in the adult. There are a number of factors involved inneoangiogenesis; these include, but are not limited to, VascularEndothelial Growth Factor (VEGF), basic Fibroblast Growth Factor (bFGF),various members of the Transforming Growth factor beta (TGFβ) family andHypoxia-Inducible Transcription Factor (HIF). Other factors that haveangiogenic properties include the Angiopoietins, (Ang-1); HepatocyteGrowth Factor (HGF); Platelet-Derived Growth Factor (PDGF); Insulin-likeGrowth Factor family (IGF-1, IGF-2) and the Neurotrophins (NGF).

The VEGFs and their corresponding receptors are key regulators in acascade of molecular and cellular events that ultimately lead to thedevelopment of the vascular system, either by vasculogenesis,angiogenesis or in the formation of the lymphatic vascular system.Although VEGF is a critical regulator in physiological angiogenesis, italso plays a significant role in skeletal growth and repair.

In the mature established vasculature, the endothelium plays animportant role in the maintenance of homeostasis of the surroundingtissue by providing the communicative network to neighboring tissues torespond to requirements as needed. Furthermore, the vasculature providesgrowth factors, hormones, cytokines, chemokines and metabolites, and thelike, needed by the surrounding tissue and acts as a barrier to limitthe movement of molecules and cells. Signals and attractant factorsexpressed on the bone endothelium help recruit circulating cells,particularly hematopoietic cells, to the bone marrow and coordinate withmetastatic cells to target them to skeletal regions. Thus, anyalteration in the vascular supply to bone tissue can lead to skeletalpathologies, such as osteonecrosis (bone death caused by reduced bloodflow to bones), osteomyelitis (infection of the bone or bone marrow bymicroorganism), and osteoporosis (loss of bone density). A number offactors have been found to have a prominent effect on the pathology ofthe vasculature and skeleton, including Osteoprotegerin (OPG), whichinhibits Receptor Activator of NF-κβ Ligand (RANKL)-inducedosteoclastogenic bone resorption.

Intramembraneous and endochondral bone ossification occur in closeproximity to vascular ingrowth. In endochondral ossification, thecoupling of chondrogenesis and osteogenesis to determine the rate ofbone ossification is dependent on the level of vascularization of thegrowth plate. For example, vascular endothelial growth (VEGF) factorisoforms are essential in coordinating metaphyseal and epiphysealvascularization, cartilage formation, and ossification duringendochondral bone development. HIF-1 stimulates transcription of theVEGF gene (and of other genes whose products are needed when oxygen isin short supply). The VEGF protein is secreted, diffuses through thetissue, and acts on nearby endothelial cells.

The response of the endothelial cells includes at least four components.First, the cells produce proteases to digest their way through the basallamina of the parent capillary or venule. Second, the endothelial cellsmigrate toward the source of the signal. Third, the cells proliferate.Fourth, the cells form tubes and differentiate. VEGF acts on endothelialcells selectively to stimulate this entire set of effects. Other growthfactors, including some members of the fibroblast growth factor family,also can stimulate angiogenesis, but they influence other cell typesbesides endothelial cells. As the new vessels form, bringing blood tothe tissue, the oxygen concentration rises, HIF-1 activity declines,VEGF production is shut off, and angiogenesis ceases.

The vascularization of cartilage regions in long bones occurs atdifferent stages of development. In early embryonic development, bloodvessels that originate from the perichondrium invaginate into thecartilage structures. During elevated postnatal growth, capillariesinvade the growth plate of long bones. In adulthood, angiogenesisperiodically can be switched on during bone remodeling in response tobone trauma or pathophysiological conditions such as rheumatoidarthritis (RA) and osteoarthritis (OA).

Bone has the unique capacity to regenerate without the development of afibrous scar, which is symptomatic of soft tissue healing of wounds.This is achieved through the complex interdependent stages of thehealing process, which mimic the tightly regulated development of theskeleton. Following trauma with damage to the musculoskeletal system,disruption of the vasculature leads to acute necrosis and hypoxia of thesurrounding tissue. This disruption of the circulation leads to theactivation of thrombotic factors in a coagulation cascade leading to theformation of a hematoma. The inflammatory response and tissue breakdownactivate factors such as cytokines and growth factors that recruitosteoprogenitor and mesenchymal cells to the fracture site. Thestimulation of the endosteal circulation in the fractured bone allowsmesenchymal cells associated with growing capillaries to invade thewound region from the endosteum and bone marrow. At the edge of a bonefracture, the transiently formed granulation tissue is replaced byfibrocartilage. Concomitantly, the periosteum directly undergoesintramembranous bone formation leading to the formation of an externalcallus; while internally, the tissue is being mineralized to form wovenbone. After stabilization of the bone tissue and vasculature in the bonefracture, the cell mediated remodeling cascade is activated whereosteoclastic removal of necrotic bone is followed by the replacement ofthe large fracture callus by lamellar bone, the callus size is reducedand the normal vascular supply is restored.

A plurality of mediators associated with fetal and postnatal bonedevelopment plays a prominent role in the cascade response in bonefracture repair. These include but are not limited to BMP-2 and 4, VEGF,bFGF, TGF-β, and PDGF. VEGF expression is detected on chondroblasts,chondrocytes, osteoprogenitor cells and osteoblasts in the fracturecallus where it is highly expressed in angioblasts, osteoprogenitor andosteoblast cells during the first seven days of healing but decreasesafter eleven days. Additionally, osteoclasts release heparinase thatinduces the release of the active form of VEGF from heparin, activatingnot only angiogenesis but also osteoclast recruitment, differentiationand activity leading to the remodeling of the fracture callus duringendochondral ossification. Fractures in some cases fail to repair orunite resulting in fibrous filled pseudarthrosis. A number ofcontributing factors can lead to non-union or delayed union of bonefractures, such as, but not limited to, anti-inflammatory drugs,steroids, Vitamin C, Vitamin D and calcium deficiencies, tobaccosmoking, diabetes, and other physiological disorders.

The absence of a functional vascular network is also an important factorin the lack of bone healing in non-union fractures. Studies havereported that angiogenic factors released from biomimetic scaffolds canenhance bone regeneration and that combination strategies that releaseboth angiogenic and osteogenic factors can enhance the regenerativecapacity of bone.

The critical sequential timing of osteoclast differentiation andactivation, angiogenesis, recruitment of osteoprogenitor cells and therelease of growth factors such as BMP-2 in osteogenesis and fracturerepair may be enhanced by the synchronized endogenous production ofangiogenic and osteogenic mediators. Studies in rat femoral drill-holeinjury have shown differential expression of VEGF splicing isoformsalong with its receptors, indicating an important role in the bonehealing process. Other studies have demonstrated that angiogenesisoccurs predominantly before the onset of osteogenesis in bonelengthening in an osteodistraction model.

Another angiogenic inducing growth factor, FGF-2, can acceleratefracture repair when added exogenously to the early healing stage of abone. Although the mechanism has not been fully elucidated, it has theability to stimulate angiogenesis and the proliferation anddifferentiation of osteoblasts to possibly aid the repair of bonefractures.

2.3. Cartilaginous Tissue Compartments

Cartilaginous tissue compartments are specialized connective tissuecompartments comprising cartilage cells, known as chondrocytes,cartilage fibers and ground substance constituting the cartilage matrix,that collectively contribute to characteristic elastic firmnessrendering cartilage capable of withstanding high levels of pressure orsheer. Cartilage is histologically classified into three types dependingon its molecular composition: hyaline cartilage; fibrocartilage andelastic cartlage.

Hyaline cartilage is the predominant form of cartilage comprising anamorphous matrix surrounding chondrocytes embedded within spaces, knownas lacunae. Hyaline cartilage, which is commonly associated with theskeletal system and found in the nose, trachea, bronchi and larynx,predominantly functions to provide support. Hyaline cartilage associatedwith the articular portions of bone, forming the major component ofsynovial joints, is termed articular cartilage. Hyaline cartilage isusually avascular except where vessels may pass through to supply othertissues and in ossification centers involved in intracartilaginous bonedevelopment.

Fibrocartilage, which is commonly found in intervertebral discs andpubic symphysis and functions to provide tensile strength and in shockabsorption, is less firm than hyaline cartilage. It comprises acombination of dense collagenous fibers with cartilage cells and a scantcartilage matrix. Fibrocartilage is not usually circumscribed by aperichondrium. Proportions of cells, fibers and ECM components infribrocartilage are variable.

Elastic cartilage, which is found in the external ear, the Eustachiantube, epiglottis and some of the lanryngeal cartilages, is characterizedby a large number of elastic fibers that branch and course in alldirections to form a dense network of anastomising and interlacingfibers.

2.3.1. Articular Cartilage Matrix

The chondrocytes in articular cartilage are surrounded by a narrowregion of connective tissue ECM, termed the pericellular matrix (PCM),which together with the chondrocyte, is termed chondron. The PCM, whichis very rich in fibronectin, proteoglycans (e.g., aggrecan, hyaluron anddecorin) and collagen (types II, VI and IX), is particularlycharacterized by a high concentration of type VI collagen as compared tothe surrounding ECM. In normal articular cartilage, type VI collagen isrestricted to the chondrons, but in osteoarthritic cartilage, it isupregulated and found throughout the ECM. A proteomic analysis ofarticular cartilage revealed the presence of collagen α1 (II)C-propeptide, collagen α1 (XI) C-propeptide, collagen α2 (XI)C-propeptide, collagen α1 (VI), collagen α2 (VI), link protein,biglycan, .decorin, osteonectin, matrillin-1, annexin-V, lactadherin,and binding immunoglobulin protein (BiP), in addition to metabolicproteins. (Wilson et. al., 2008, Methods, 48: 22-31).

2.3.2. Chondrocyte Differentiation

The specific structure of articular cartilage, with endogenouschondrocytes forming adult joints, is the result of endochondralossification, as described above under the Heading, Osseous TissueCompartments Formation.

Chondrocyte differentiation and maintenance in articular cartilage isgoverned by interaction of multiple factors. Key players include, butare not limited to, ions (e.g., calcium); steroids (e.g., estrogens);terpenoids (e.g., retinoic acid); peptides (e.g., Parathyroid hormone(PTH), parathyroid hormone-related peptide (PTHrP)), insulin growthfactors (e.g., TGFβ hormones, including, without limitation, BMPs,IGF-1, VEGF, PDGF, FGF); transcription factors (e.g., Wnt, SOX-9);eicosanoids (e.g., prostaglandins); catabolic interleukins (e.g., IL-1);and anabolic interleukins (e.g., IL-6, IL-4 and IL-10). (Gaissmaier etal., 2008, Int. J. Care Injured, 39S1: S88-S96).

2.3.3. Growth Plate

The epiphyseal plates or growth plates are a hyaline cartilage platelocated in the metaphysis at the end of long bones. Whereas endochondralossification is responsible for the formation of cartilage in utero andin infants, the growth plates are responsible for the longitudinalgrowth of long bones via a cartilage template. The ongoing developmentalprocesses of proliferation and differentiation within the growth platesare mediated by a number of hormonal and paracrine factors secreted bythe growth plate chondrocytes. The growth plate is a highly organizedstructure comprising a large number of chondrocytes in various stages ofdifferentiation and proliferation embedded in a scaffold of ECMcomponents.

The growth plate can be subdivided into four zones depending on thestage of differentiation and spatial distribution of collagen types. Theresting zone is the smallest zone close to the epiphyseal cartilagecomprising small monomorphic chondrocytes with a narrow rim ofcytoplasm. The chondrocytes of the resting zone secrete growth plateorienting factor (GPOF) that aligns proliferating cells parallel to thelong axis of the developing bone. Stem cell-like cells of the restingzone have a limited proliferative capacity, which eventually leads tofusion of the growth plate (epiphyseal fusion). The proliferative zoneof the growth plate comprises chondrocytes that are arranged incharacteristic columns parallel to the longitudinal axis of the bone andare separated by ECM with high type II collagen. The chondrocytes of theproliferative zone are mitotically active, have high oxygen and glycogencontent, and exhibit increased mitochondrial ATP production. Thehypertrophic zone refers to the zone farthest from the resting zonewhere prehypertrophic chondrocytes stop dividing and terminallydifferentiate into elongated hypertrophic chondrocytes embedded in ECMhigh in type X collagen. Hypertrophic chondrocytes have a highintracellular calcium concentration required for the production ofrelease vesicles containing Ca2+-binding annexins, that secrete calciumphosphate, hydroxyapatite, phosphatases (such as alkaline phosphatase),metalloproteinases, all instrumental in proteolytic remodeling andmineralization of the surrounding matrix. The hypertrophic chondrocytesproduce factors, such as VEGF, that initiate vascularization of themineralized matrix that is then degraded by invading phagocyticchondroclasts and osteoclasts constituting the invading zone.

The developmental processes involving chondrogenesis are regulated by aninterplay of a large number of systemic hormones and paracrine factors,including growth factors, cytokines and transcription factors. Keyfactors involved in chondrocyte proliferation and differentiation in thegrowth plate including ATF-2, BCL-2, Inn, PTHrP, BMP, PGE2, MMP, Sox,Runx2 (Cbfal), NOTCH, HOX, FGF are reported by Brochhausen et al. (J.Tissue Eng. Regen. Med. 2009, 3: 416-429).

2.3.4. Stem Cells of Cartilaginous Tissue Compartments

Multipotent mesenchymal progenitor cells with adipogenic, osteogenic andchondrogenic potential, and that are CD105+/CD166+(corresponding toTGF-β type III receptor (endoglin) and ALCAM, respectively), have beenidentified in articular cartilage. (Asalameh et al., Arthritis &Rheumatism, 2004, 50(5): 1522-1532). The presence ofCD34−/CD45−/CD44+/CD73+/CD90+ mesenchymal stem cells with adipogenic,chondrogenic and osteogenic potential also has been shown. (Peng et al.,Stem Cells and Development (2008), 17: 761-774). Similar to bone-derivedMSCs, articular-derived MSCs are positive for surface expression ofNotch-1. (Hiraoka et al., Biorheology, 2006, 43: 447-454). A potentialMSC niche positive for Stro-1, Jagged-1 and BMPr1a has also beenidentified in the perichondrial zone of Ranvier on the growth plate.(Karlsson et al., 2009, J. Anat. 215(3): 355-63).

Differential expression of Notch-1, Stro-1 and VCAM-1/CD106 markers hasbeen observed in normal articular cartilage versus osteoarthritic (OA)cartilage. In normal cartilage, expression of these markers is higher inthe superficial zone (SZ) as compared to the middle zone (MZ) and deepzone (DZ). On the other hand, OA cartilage SZ has reduced Notch-1 andSox-9 while MZ has increased Notch-1, Stro-1 and VCAM-1 positive cells.(Grogan et al., Arthritis Res. Ther. 2009, 11(3): R85-R97).

2.3.5. Intervertebral Disc Fibrocartilage Tissue Compartments

The intervertebral discs (IVD) predominantly are comprised offibrocartilage. The IVD fibrocartilage is continuous both with and belowthe articular cartilage of adjacent vertebrae as well as peripherallywith spinal ligaments. The IVD is a unique structure containing annulusfibrosus (AF) and nucleus pulposus (NP), a gelatinous ellipsoidalremnant of the embryonic notochord, and is sandwiched between twoadjacent cartilaginous endplates (EP). IVD rupture and herniation of thenucleus pulposus into the spinal cord may cause severe pain and otherneurological symptoms. The NP and AF synergistically function to achievethe primary role of IVD in transferring load, dissipating energy andfacilitating in joint mobility.

The adult IVD is essentially avascular; hence, endogenous cells survivein a low-nutrient and low-oxygen microenvironment. The major ECMcomponents of IVD include but are not limited to aggrecan, collagen(e.g., types I, II and IX), leucine rich repeat (LRR) proteins andproteoglycans (e.g., fibromodulin, decorin, lumican), cartilageoligomatrix protein, and collagen VI beaded filament network. (Feng etal., 2006, J. Bone Joint Surg. Am. 88: 25-29). The water content, GAGcontent, aggrecan levels and levels of type II collagen aresignificantly lower in older discs demonstrating the effects of IVDdegeneration with age. (Murakami et al., 2010, Med. Biol. Eng. Comput.48: 469-474).

The central nucleus pulposus (NP) is rich in aggrecan and hyaluron. Thedeveloping NP is characterized by the presence of highly vacuolatedchondrocytes and small chondroblasts inherited from the notochord.Primarily functioning as a primitive axial support, the integrity of thenotochord is maintained by a proteoglycan (PG-) and laminin-rich sheath.As NP matures, the cellular composition becomes predominantlychondrocytic. Mature NP cells are small and have an aggrecan richmatrix, which is essential in maintaining requisite hydration levels formechanical function. Their gene expression profile and metabolicactivity are distinct from the chondrocytes of articular cartilage. TheECM of immature NP has high aggrecan levels and primarily contains typeII collagen, with the type IIA isoform expressed by progenitor cellsduring chondrogenesis, not by mature chondrocytes. (Hsieh A. H. andTworney J. D., J. Biomech., 2010, 43(1): 137-156).

The AF surrounds the NP with layers of unidirectional sheets of collagenparallel to the circumference of a disc to form collagen lamellae.Alternating bidirectional collagen fibers intersperse the AF collagenlamellae. AF can be subdivided into three regions: inner AF, middle AFand outer AF. The inner AF arises along with endochondral formation ofthe vertebrae. The outer AF arises as a separate cell condensation withslower matrix formation. Lamellae of inner AF comprises predominantly oftype II collagen and fibrochondrocytes, while those of outer AF arecomprised of type I collagen and fibroblasts. A population of pancakeshaped interlamellar cells as well as elastin fibers are also foundwithin the lamellae, in vertebral attachments, and at the NP-AFinterface. Large proteoglycans (PGs; for example aggrecan and versican)and type I and VI collagen permeate interlamellar and translamellar ECM.(Hsieh A. H. and Tworney J. D., J. Biomech., 2010, 43(1): 137-156).

A large number of coordinated signals originating from the cells of thenotochord and floor plate of the embryonal neural tube are instrumentalin disc embryogenesis. Key signals include, but are not limited to,sonic hedgehog (Shh), Wnt, noggin, Pax family of transcription factors(e.g., Pax 1 and Pax 9), Sox family of transcription factors (Sox5, Sox6and Sox) and TGF-β. (Smith et al., 2011, Dis Model Mech. 4(1): 31-41).Herniation and IVD degeneration are associated with changes ininflammatory and immune cytokine profiles, including, but not limitedto, the activation of Th1-related cytokines (e.g. IFNγ) as well asTh17-related cytokines (e.g., IL-4, IL-6, IL-12 and IL-17). (Shamji etal., 2010, Arthritis & Rheumatism, 62(7): 1974-1982).

A potential stem cell niche comprised of progenitor cells that arepositive for Notch1, Delta4, Jagged1, CD117, Stro-1 and Ki67 has beenidentified in intervertebral discs of a number of animals, includinghumans. It has been reported that the IVD tissue compartments comprise aslow growing zone in the AF as well as the NP regions. (Henriksson etal., 2009, SPINE, 34(21): 2278-2287).

2.4. Cardiovascular Tissue

Cardiovascular tissues can include the tissue that comprises any organinvolved in the cardiovascular function of an organism. The cardiovascular system permits blood to circulate and transport nutrients (suchas amino acids and electrolytes), oxygen, carbon dioxide, hormones, andblood cells to and from the cells in the body to provide nourishment andhelp in fighting diseases, stabilize temperature and pH, and maintainhomeostasis. Tissues of the cardiovascular system can include arteries,capillaries, veins, coronary vessels, portal veins, and the heart,including all of its associated structures. Other types of cardiactissue can include connective tissue, and cardiac muscle. Cardiac muscleis comprised of the epicardium, myocardium, and endocardium. Theepicardium is the outer layer of cardiac muscle. The myocardium is thethick middle layer of cardiac muscle. The endocardium is the inner mostportion of cardiac muscle.

2.5. Cornea Tissue

Cornea tissue refers to the transparent front facing structure of theeye, which cover the iris, pupil, and anterior chamber. The cornearefracts light with the anterior chamber and the lens and is responsiblefor the optical power of the eye. The human cornea has five layersincluding the corneal epithelium, anterior limiting membrane (Bowman'slayer), substantia propria (Corneal stroma), posterior limiting membrane(Descemet's membrane), and the Corneal endothelium.

2.6. Dental Tissue Compartments

A tooth has three anatomical divisions (crown, root and neck), and fourstructural components (enamel, dentin, cementum and pulp).

Enamel is the hardest, most mineralized biological tissue in the humanbody. It is composed of elongated hydroxyapatite crystallites bundledinto rods or prisms, interspersed with crystalline interrods filling theinterstitial space. Enamel cells, known as ameloblasts, are responsiblefor enamel development. Ameloblastin, TRAP and enamelin are key proteinsfound in enamel tissue whereas the enamel matrix is devoid of collagen,composed primarily of amelogenin. An intricate orchestration ofsignaling factors, such as BMPs (e.g., BMP-2, BMP-4, BMP-7), FGFs (e.g.,FGF-3, -4, -9, -20), Wnt-3, 10a, 10b and transcription factors, such as,p21, Msx2 and Lef1 is responsible for morphogenesis of enamel.Self-assembly of amelogens to form amelogenin nanospheres play a role innucleation of hydroxyapatite crystallization and enamel mineralization.Matrix processing enzymes, such as MMP-20, kallikrein-4 (KLK4), alsoknown as enamel matrix serine protease-1 (EMSP-1), are involved in thecomplete elimination of the protein matrix and replacement with amineralized matrix. (Fong et al., 2005, J. Dent. Educ., 69(5): 555-570).Ameloblasts arise from epithelial stem cells of ectodermal origin. Theyare lost after tooth eruption leaving no adult human ectodermal stemcells in the mature enamel. In contrast, rodent enamel retain a niche ofepithelial stem cells, known as apical bud cells, for continuous enamelproduction. (Ulmer et al., 2010, Schweiz Monatsschr Zahnmed,120:860-872).

Dentin is a hard, yellowish and elastic living connective tissuecompartment with biomechanical properties similar to bone. The formationof dentin is driven by mesenchymally derived mature odontoblasts thatare fully differentiated and nondividing and that form a single layerunderneath the dentin in a mature tooth. A series ofepithelial-mesenchymal interactions regulates odontoblastdifferentiation from neural crest cells in the first branchial arch andfrontonasal processes. Mature dentin is comprised of a mantle, composedof intertubular and peritubular dentin made of a collagen fibril matrix,with odontoblast cell processes extending into dentin tubules. Duringdentinogenesis, odontoblasts secrete predentin, a mineralized tissuecomposed of type I collagen. Unlike osteogenesis, in dentinogenesis, asthe predentin layer is formed, the odontoblasts recede instead ofbecoming embedded within the dentin matrix, leaving behind cellsprocesses within dentinal tubules. Subsequently, the unmineralizedpredentin is converted to dentin by gradual mineralization of collagen.Dentinogenesis is directed by a series of highly controlled biochemicalevents that control the rates of collagen secretion, its maturation intothick fibrils, loss of proteoglycans, mineral formation includinghydroxy apatite crystallization, and growth. The dentin matrix isprimarily composed of collagens (e.g., types I, III and V) as well asother matrix proteins, including, but not limited to, phosphorylated andnonphosphorylated matrix proteins, proteoglycans, growth factors,metalloproteinases, alkaline phosphatase serum derived proteins, andphospholipids. (Fong et al., 2005, J. Dent. Educ., 69(5): 555-570). Nostem cells have been identified in mature dentin.

The dental pulp is the tooth's living tissue that respond to pain anddamage and initiates tissue repair. An odontoblast cell layer forms theouter boundary of the pulp and is associated with an underlying networkof dendritic cells. A cell-free zone underlying the odontoblast layer isrich in nerve fibers and blood vessels. Similar to dentin, dental pulpalso differentiates from neural crest-derived ectomesenchyme duringtooth development.

Several sources of stem cells have been identified associated with pulptissue. In immature teeth, apical papilla, the embryonal organresponsible for pulp differentiation, is the source for stem cells ofapical papilla (SCAP). Mature dental pulp is the source of dental pulpstem cells (DPSC) whereas stem cells are also extracted from exfoliateddeciduous teeth (SHED) Additional cells of the dental pulp core thatfunctionin pulpal defense, include, but are not limited to, macrophages,lymphocytes and mast cells. Pulp matrix is composed of collagens (e.g.,types I, III V and VI), but lacks mineralization. Other noncollagenousproteins of the pulp matrix are similar in composition to dentin. Thedental pulp is capable of responding to dentin tissue damage bysecreting new dentin from old odontoblast populations or generation andsecretion of dentin from new secondary odontoblast populations. (Fong etal., 2005, J. Dent. Educ., 69(5): 555-570).

The periodontium consists of tissues supporting the tooth crown,including a nonmineralized periodontal ligament (PDL) sandwiched betweenlayers of mineralized tissues, including the cementum, alveolar bone anddentin. Cementum is a thin mineralized layer covering the dentin.Cementoblasts are cells responsible for cementum matrix secretion andsubsequent mineralization. When cementoblasts become entrapped withincementum matrix, they are termed cementocytes. Cementoblasts areectomesenchymal, being derived from neural crest cells, similar to PDLand alveolar bone. Like bone and dentin, cementum is a collagenousmineralized tissue that hardens upon formation of carbonatedhydroxyapatite. (Fong et al., 2005, J. Dent. Educ., 69(5): 555-570).

PDL is a space between cementum and alveolar bone. It represents areplacement of the dental follicle region in immature developing teeth.Mature PDL contains mostly periodontal fibroblasts as well as stemcells, known as the periodontal ligament stem cells (PDLSCs). Theimmature dental follicle is also a source of mesenchymal stem cells,known as dental follicle stem cells (DFSCs). (Fong et al., 2005, J.Dent. Educ., 69(5): 555-570). The differentiation potential of dentalmesenchymal cells has been reviewed by Ulmer et al., Zahnmed, 2010,Schweiz Monatsschr 120:860-872 and is incorporated herein by referencein its entirety.

Several dental stem cell markers have been identified. Stro-1 and Stro-4are commonly used dental stem cell markers for all dental mesenchymalstem cells. Dental stem cells originating from the neural crest have theneural marker, nestin. An osteoblast marker, osteocalcin, is also usedas a stem cell marker for DPSCs. Similarly, SCAPs express Oct-4, Nanog,SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. (Ulmer et al., 2010, SchweizMonatsschr Zahnmed, 120:860-872).

2.7. Fascial Tissue Compartment

Fascial tissue compartments form a layer of fibrous tissue foundthroughout the body surrounding softer and more delicate organs,including but not limited to muscles, groups of muscles, blood vessels,nerves, etc. Fascial tissue originates from the embryonic mesenchyme.Fasciae form during the development of bones, muscles and vessels fromthe mesodermal layer of the embryo. Fascial tissue can be categorizedinto three types depending on location: (1) superficial fascial tissue,which is found beneath the integument throughout the body, usuallyblending with the reticular layer of the dermis; (2) deep fascial tissuecomprising dense fibroareolar connective tissue surrounding muscles,bones, nerves and blood vessels; and (3) visceral or subserous fascia,which suspends organs within their cavities and wraps them in layers ofconnective tissue membranes. (Chapter IV. Myology, Section 3. Tendons,Aponeuroses, and Fasciae, Gray's Anatomy of the Human Body, 20thEdition, Re-edited by Lewis, W. H., Lea & Febiger, Philadelphia, 1918,Bartleby.com, New York, 2000).

The fibroareolar connective tissue of fascia comprises four kinds ofcells: (1) flattened lamellar cells, which may be branched or unbranched(branched lamellar cells contain clear cytoplasm and oval nuclei andproject multidirectional processes that may unite to form an opennetwork, such as in the cornea; unbranched lamellar cells are joined endto end. (2) Clasmatocytes, which are large irregular vacuolated orgranulated cells with oval nuclei. (3) Granule cells, which are ovoid orspherical in shape. (4) Plasma cells of Waldeyer, usually spheroidal,characterized by vacuolated protoplasm.

2.8. Hair Follicles

Hair follicles are the mammalian skin organ responsible for theproduction of hair. Hair follicles are comprised of several structuresincluding the papilla, matrix, root sheath, bulge, and other supportingstructures including the fundibulum, the arrector pili muscles, thesebaceous glands, and the apocrine sweat glands. Hair follicle receptorssense the position of the hair. The papilla is a large structuresituated at the base of the hair follicle, made up of mainly ofconnective tissue, and a capillary loop. The area surround the papillais defined by hair matrix. The root sheath is composed of two portions:an external and internal root sheath. The external root sheath appearsempty with cuboid cells. The internal root sheath is composed of threelayers, Henle's layer, Huxley's layer, and an internal cuticle, which iscontinuous with the outermost layer of the hair fiber.

2.9. Ligament Tissue Compartment

The term “ligaments” as used herein refers to dense regular connectivetissue comprising attenuated collagenous fibers that connect bones atjoints. Ligament ECM is composed of type I and type III collagenstogether with other proteoglycans and glycoproteins. Mesenchymal stemcells have been found in the human anterior cruciate ligament thatexhibit multilineage differentiation potential, like bone-derivedmesenchymal stem cells. (Cheng et al., 2010, Tissue Engg. A,16(7):2237-2253).

2.10. Meniscus

The meniscus is comprised of two pads or menisci comprised offibrocartilaginous tissue which serve to disperse friction in the kneejoint between the tibia (lower leg) and femur (the thigh). The menisciare concave on the top portion and flat on the bottom portion,articulating with the tibia. The menisci are attached to the fossae(small depressions) between the condyles of the tibia (intercondyloidfossa), and towards the center they are unattached and their shapenarrows to a thin shelf. The menisci act to disperse the weight of abody and reduce friction during movement.

2.11. Reproductive Tissue and Foreskin

The reproductive tissues can include the tissues that comprise any organinvolved in the reproductive process of an organism. Vertebrate animalsall share the key elements of their reproductive systems, includinggamete producing organs or gonads, which can be broken down to male andfemale counterparts.

The major reproductive organs of the male can be grouped into threecategories. 1. Sperm production and storage. Production takes place inthe testes which are housed in the temperature regulating scrotum,immature sperm then travel to the epididymis for development andstorage. 2. Ejaculatory fluid producing glands which include the seminalvesicles, prostate, and the vas deferens. 3. Organs used for copulation,and deposition of the spermatozoa (sperm) within the male, these includethe penis, urethra, vas deferens, and Cowper's gland. The foreskin is adouble-layered fold of smooth muscle tissue, blood vessels, neurons,skin, and mucous membrane that covers and protects the glans penis andthe urinary meatus. The foreskin can also be described as the prepuce, atechnically broader term that also includes the clitoral hood in women,to which the foreskin is embryonically homologous.

The male reproductive system can include the following structures. Thetestes can include: Tunica vaginalis, Tunica albuginea, Tunicavasculosa, Appendix Mediastinum, Lobules Sept, Leydig cells, and Sertolicells. Internal structures critical to the function of the malereproductive system can include: Seminiferous tubules (e.g., Tubuliseminiferi recti, Rete testis, Efferent ducts), Epididymis- (e.g.,Appendix and Stereocilia), Paradidymis Spermatic cord, Vas deferensAmpulla, and Ejaculatory duct. Accessory glands of the male reproductivesystem can include: Seminal vesicles (e.g., excretory duct), Prostate(e.g., Urethral crest), Seminal colliculus, Prostatic utricle,Ejaculatory duct, Prostatic sinus, Prostatic ducts, and Bulbourethralglands. The penis can include the following structures: root (e.g., CrusBulb, Fundiform ligament, Suspensory ligament), body (e.g., Corpuscavernosum, Corpus spongiosum), glans (e.g., Foreskin, Frenulum Corona),fascia (e.g., superficial deep), Tunica albuginea, and Septum. Theurinary tract can include: Internal urethral orifice, Urethra (e.g.,Prostatic, Intermediate, Spongy), Navicular fossa, External urethralorifice, Lacunae of Morgagni, and Urethral gland. The scrotum caninclude the skin layers (e.g., Dartos, External spermatic fascia,Cremaster, Cremasteric fascia, Internal spermatic fascia), Perinealraphe and Scrotal septum.

The human female reproductive system is a series of organs primarilylocated inside of the body and around the pelvic region of a female thatcontribute towards the reproductive process. The human femalereproductive system contains three main parts: the vagina, which leadsfrom the vulva, the vaginal opening, to the uterus; the uterus, whichholds the developing fetus; and the ovaries, which produce the female'sova. The breasts are involved during the parenting stage ofreproduction, but in most classifications they are not considered to bepart of the female reproductive system. The vagina meets the outside atthe vulva, which also includes the labia, clitoris and urethra; duringintercourse this area is lubricated by mucus secreted by the Bartholin'sglands. The vagina is attached to the uterus through the cervix, whilethe uterus is attached to the ovaries via the fallopian tubes. Eachovary contains hundreds of egg cells.

The female reproductive system can include the following structures. Theovaries can include the: corpus (e.g., hemorrhagicum, luteum, albicans),Theca of follicle (e.g., externa and interna), Follicular antrum,Follicular fluid, Corona radiata, Zona pellucida, Membrana granulosa,Perivitelline space, Germinal epithelium, Tunica albuginea, cortex(e.g., Cumulus oophorus and Stroma), and Medulla. The fallopian tubescan include the: Isthmus, Ampulla, Infundibulum, Fimbria, and Ostium.Ovary ligaments can include the: Proper of ovary and Suspensory ofovary. Wolffian vestiges can include the: Gartner's duct, Epoophoron,(e.g., Vesicular appendages of epoophoron) and Paroophoron.

The uterus can include the following regions: corpus/body (e.g., Uterinecavity, Fundus), Cervix (e.g., External orifice, Canal, Internalorifice, Supravaginal portion, Vaginal portion), and Uterine horns. Theuterus layers can include the: Endometrium, Myometrium, Perimetrium andParametrium. The uterus can include glands, for example, the utuerinegland. The uterus can include ligaments including the: Round ligament,Broad ligament, Cardinal ligament, Uterosacral ligament and Pubocervicalligament. The vagina can include the: Fossa of vestibule of vagina,vaginal fornix and Hymen. The Labia can include the: Mons pubis, Labiamajora (e.g., Anterior commissure and Posterior commissure), Pudendalcleft, Labia minora (e.g., Frenulum of Labia minora and Frenulum ofclitoris), Vulval vestibule, Interlabial sulci, Bulb of vestibule,Vaginal orifice, vestibular glands/ducts (e.g., Bartholin'sglands/Bartholin's ducts, Skene's glands/Skene's ducts). The clitoriscan include the: Crus of clitoris, Corpus cavernosum, clitoral glans,Hood. The urethra can include the urethral crest. Other femalereproductive structures can include the: Grafenberg spot, Urethralsponge and Perineal sponge.

2.12. Spinal Disc

Spinal disc can otherwise be known as intervertebral disc or theintervertebral fibrocartilage. The term spinal disc refers to tissuewhich lies between adjacent vertebrae in the vertebral column. Eachspinal disc forms a fibrocartilaginous joint, thereby allowing movementof the vertebrae. Spinal discs also act as a ligament to hold thevertebrae together. Spinal disc role as shock absorbers in the spine iscrucial.

Spinal disc consist of an outer fibrous ring, (e.g., the anulusfibrosus), which surrounds an inner gel-like center (e.g., the nucleuspulposus). The anulus fibrosus consists of several layers or laminae offibrocartilage, made up of both type I and type II collagen. Type Icollagen is concentrated towards the edge of the ring where it providesgreater strength. The stiff laminae can withstand compressive forces.The fibrous intervertebral disc contains the nucleus pulposus, whichassist in the distribute pressure evenly across the disc, preventing thedevelopment of stress concentrations which could cause damage to theunderlying vertebrae or to their endplates. The nucleus pulposuscontains loose fibers that are suspended in a mucoprotein gel. Thenucleus pulposus acts as a shock absorber, absorbing the impact of thebody's activities and keeping the two vertebrae separated.

2.13. Synovial Tissue Compartment

The synovial membrane is composed of fibrous connective tissue and linesthe joint cavity of synovial joints. It is made up of a layer ofmacrophage (type A) and fibroblast-like (type B) synoviocytes and aloose sublining tissue. Synovial fluid is secreted by synovial cellslining the synovial membrane in the joint capsule. It is a viscid,mucoalbuminous fluid, rich in hyaluronic acid. It acts as a lubricatingfluid, facilitating the smooth gliding of the articular surface.Functional mesenchymal stem cell niches have been identified as residentto synovial lining and subsynovial tissue. These cells are positive forthe artificial nucleoside, iododeoxyuridine (IdU) as well as MSC markerssuch as PDGFRα, p75 and CD44 and have chondrogenic potential. (Kurth etal., Arthritis Rheum., 2011, 63(5): 1289-1300). Synovial fluid-derivedMSCs have also been identified, and these have higher chondrogenicpotential as compared to bone marrow-derived and adipogenic MSCs. (Kogaet al., 2008, Cell Tissue Res., 333: 207-215). Synovial MSCs and MPCshave been shown to prevent degeneration due to intervertebral discdisease (IVD) and to be useful for cartilage tissue engineering.(Miyamoto et al., 2010, Arthritis Res. Ther., 12: R206-218; Lee et al.,2010, Tissue Engg. A, 16(1): 317-325).

2.14. Tendon Tissue Compartment

Tendons are specialized connective tissue compartments that connect boneto muscle. Tendon cells are embedded amongst a parallel group ofcollagenous fibers that secrete a unique ECM containing collagens, largeproteoglycans, and small leucine rich proteoglycans that function aslubricators and organizers of collagen fibril assembly. A unique tendonstem/progenitor cell (TSPC) niche has been identified amongst theparallel collagen fibrils surrounded by ECM. The TSPCs exhibitosteogenic and adipogenic potential. Biglycan and fibromodulin are keytendon ECM components that direct TSPC fate through BMP signaling. TheseTSPCs are positive for bone marrow derived stem cell markers such asStro-1, CD146, CD90 and CD44 but not for CD18. TSPCs do not expresshematopoietic markers, such as CD34, CD45 and CD117, or the endothelialmarker CD106. (Bi et al., 2007, Nat. Med., 13(10): 1219-1227).

2.15. Vasculature Tissue Compartment

The vascular wall is made of three concentric zones with distinctcellular composition, all mesodermal in origin: the tunica intima,containing predominantly mature differentiated endothelial cells (EC),the tunica media, containing mature and differentiated smooth musclecells, and the tunica adventitia, containing mature fibroblasts. (Tilkiet al., 2009, Trends Mol. Med. 15(11): 501-509). Endothelial progenitorcells (EPCs), meaning cells that exhibit clonal expression, stemnesscharacteristics, adherence to matrix molecules and an ability todifferentiate into endothelial cells (ECs) have been implicated in theformation of new blood vessels through angiogenesis and postnatalvasculogenesis. EPCs have many characteristic cell surface markers,including, but not limited to, CD34, AC133, KDR (VEGFR-2), Tie-2 andligand for UEA-1 lectin. (Tilki et al., 2009, Trends Mol. Med. 15(11):501-509; Melero-Martin and Dudley, 2011, Stem Cells, 29: 163-168;Pascilli et al., 2008, Exp. Cell Res., 315: 901-914).

EPC niches have been identified in the bone-marrow, peripheral cordblood and vascular wall matrix. Bone-marrow derived and cord blood EPCsessentially may be proangiogenic hematopoietic progenitor cells (HPCs),circulating in the blood and committed to myeloid lineage. (Tilki etal., 2009, Trends Mol. Med. 15(11): 501-509). The vascular wall stem andprogenitor cells (VW-EPCs) reside in distinct zones of the vessel wallwithin subendothelial space, known as avasculogenic zone, within thevascular adventitia, forming vascular wall-specific niches. Fetal andadult arterial and venous blood vessel walls have also been found toharbor resident niches for a variety of stem and progenitor cells, suchas EPCs, smooth muscle progenitors, HSCs, MSCs, mesangial cellscoexpressing myogenic and endothelial markers, neural stem cells (NSCs),etc. (Tilki et al., 2009, Trends Mol. Med. 15(11): 501-509). The VW-EPCsare CD34(+)VEGFR-2(+)Tie-2(+)CD31(−)CD144(−). Proliferating anddifferentiating VW-EPCs become CD144(+).

During embryogenesis, there is evidence of the existence of ahemangioblast (giving rise to endothelial and hematopoietic cells) andhemogenic endothelium, originating from precursors resident in thevascular wall. However, whether adult VW also contains ancestralprogenitor hemangioblasts giving rise to both VW-EPCs as well as VW-HSCsis not known. Vascular wall also contains resident pericyte-like cellsin the subendothelial spaces. These pericyte-like cells serve as acellular reservoir for VW-MSCs, which can differentiate into colonieswith adipogenic, osteogenic and chondrgenic markers. (Tilki et al.,2009, Trends Mol. Med. 15(11): 501-509).

Exemplary factors secreted by vascular tissue cells are disclosed inTilki et al., 2009, Trends Mol. Med. 15(11): 501-509, the entirecontents of which are incorporated herein by reference.

2.16. Placenta

The placenta is considered one of the most important sources of stemcells, and has been studied extensively. It fulfills two main desiderataof cell therapy: a source of a high as possible number of cells and theuse of non-invasive methods for their harvesting. Their highimmunological tolerance supports their use as an adequate source in celltherapy (Mihu, C. et al., 2008, Romanian Journal of Morphology andEmbryology, 2008, 49(4):441-446).

The fetal adnexa is composed of the placenta, fetal membranes, andumbilical cord. The term placenta is discoid in shape with a diameter of15-20 cm and a thickness of 2-3 cm. The fetal membranes, amnion andchorion, which enclose the fetus in the amniotic cavity, and theendometrial decidua extend from the margins of the chorionic disc. Thechorionic plate is a multilayered structure that faces the amnioticcavity. It consists of the following structures: the amniotic membrane(composed of epithelium, compact layer, and amniotic mesoderm), thechorion (composed of mesenchyme and a region of extravillousproliferating trophoblast cells interposed in varying amounts ofLanghans fibrinoid, either covered or not by syncytiotrophoblast), andthe intermediate spongy layer between the amniotic membrane and thechorion.

Villi originate from the chorionic plate and anchor the placenta throughthe trophoblast of the basal plate and maternal endometrium. From thematernal side, protrusions of the basal plate within the chorionic villiproduce the placental septa, which divide the parenchyma into irregularcotyledons (Parolini, O. et al., 2008, Stem Cell, 2008, 26:300-311).

Some villi anchor the placenta to the basal plate, whereas othersterminate freely in the intervillous space. Chorionic villi present withdifferent functions and structure. In the term placenta, the stem villishow an inner core of fetal vessels with a distinct muscular wall andconnective tissue consisting of fibroblasts, myofibroblasts, anddispersed tissue macrophages (Hofbauer cells). Mature intermediate villiand term villi are composed of capillary vessels and thin mesenchyme. Abasement membrane separates the stromal core from an uninterruptedmultinucleated layer, called the syncytiotrophoblast. Between thesyncytiotrophoblast and its basement membrane are single or aggregatedLanghans cytotrophoblastic cells, commonly called cytotrophoblast cells(Parolini, O. et al., 2008, Stem Cell, 2008, 26:300-311).

Four regions of fetal placenta can be distinguished: an amnioticepithelial region, an amniotic mesenchymal region, a chorionicmesenchymal region, and a chorionic trophoblastic region.

2.17. Amnion and Chorion

2.17.1. Amniotic Membrane

Fetal membranes continue from the edge of the placenta and enclose theamniotic fluid and the fetus. The amnion is a thin, avascular membranecomposed of an inner epithelial layer and an outer layer of connectivetissue that, and is contiguous, over the umbilical cord, with the fetalskin. The amniotic epithelium (AE) is an uninterrupted, single layer offlat, cuboidal and columnar epithelial cells in contact with amnioticfluid. It is attached to a distinct basal lamina that is, in turn,connected to the amniotic mesoderm (AM). In the amniotic mesodermclosest to the epithelium, an acellular compact layer isdistinguishable, composed of collagens I and III and fibronectin. Deeperin the AM, a network of dispersed fibroblast-like mesenchymal cells andrare macrophages are observed. It has been reported that the mesenchymallayer of amnion indeed contains two subfractions, one having amesenchymal phenotype, also known as amniotic mesenchymal stromal cells,and the second containing monocyte-like cells.

2.17.2. Chorionic Membrane

A spongy layer of loosely arranged collagen fibers separates theamniotic and chorionic mesoderm. The chorionic membrane (chorion leave)consists of mesodermal and trophoblastic regions. Chorionic and amnioticmesoderm are similar in composition. A large and incomplete basal laminaseparates the chorionic mesoderm from the extravillous trophoblastcells. The latter, similar to trophoblast cells present in the basalplate, are dispersed within the fibrinoid layer and expressimmunohistochemical markers of proliferation. The Langhans fibrinoidlayer usually increases during pregnancy and is composed of twodifferent types of fibrinoid: a matrix type on the inner side (morecompact) and a fibrin type on the outer side (more reticulate). At theedge of the placenta and in the basal plate, the trophoblastinterdigitates extensively with the decidua (Cunningham, F. et al., Theplacenta and fetal membranes, Williams Obstetrics, 20th ed. Appleton andLange, 1997, 95-125; Benirschke, K. and Kaufmann, P. Pathology of thehuman placenta. New York, Springer-Verlag, 2000, 42-46, 116, 281-297).

2.17.3. Amnion-Derived Stem Cells

The amniotic membrane itself contains multipotent cells that are able todifferentiate in the various layers. Studies have reported theirpotential in neural and glial cells, cardiac repair and also hepatocytecells. Studies have shown that human amniotic epithelial cells expressstem cell markers and have the ability to differentiate toward all threegerm layers. These properties, the ease of isolation of the cells, andthe availability of placenta, make amniotic membrane a useful andnoncontroversial source of cells for transplantation and regenerativemedicine.

Amniotic epithelial cells can be isolated from the amniotic membrane byseveral methods that are known in the art. According to one such method,the amniotic membrane is stripped from the underlying chorion anddigested with trypsin or other digestive enzymes. The isolated cellsreadily attach to plastic or basement membrane-coated culture dishes.Culture is established commonly in a simple medium such as Dulbecco'sModified Eagle's Medium (DMEM) supplemented with 5%-10% serum andepidermal growth factor (EGF), in which the cells proliferate robustlyand display typical cuboidal epithelial morphology. Normally, 2-6passages are possible before proliferation ceases. Amniotic epithelialcells do not proliferate well at low densities.

Amniotic membrane contains epithelial cells with different surfacemarkers, suggesting some heterogeneity of phenotype. Immediately afterisolation, human amniotic epithelial cells express very low levels ofhuman leukocyte antigen (HLA)-A, B, C; however, by passage 2,significant levels are observed. Additional cell surface antigens onhuman amniotic epithelial cells include, but are not limited to,ATP-binding cassette transporter G2 (ABCG2/BCRP), CD9, CD24, E-cadherin,integrins α6 and β1, c-met (hepatocyte growth factor receptor),stage-specific embryonic antigens (SSEAs) 3 and 4, and tumor rejectionantigens 1-60 and 1-81. Surface markers thought to be absent on humanamniotic epithelial cells include SSEA-1, CD34, and CD133, whereas othermarkers, such as CD117 (c-kit) and CCR4 (CC chemokine receptor), areeither negative or may be expressed on some cells at very low levels.Although initial cell isolates express very low levels of CD90 (Thy-1),the expression of this antigen increases rapidly in culture (Miki, T. etal., Stem Cells, 2005, 23: 1549-1559; Miki, T. et al., Stem Cells, 2006,2: 133-142).

In addition to surface markers, human amniotic epithelial cells expressmolecular markers of pluripotent stem cells, including octamer-bindingprotein 4 (OCT-4) SRY-related HMG-box gene 2 (SOX-2), and Nanog (Miki,T. et al., Stem Cells, 2005, 23: 1549-1559). Previous studies also haveshown that human amnion cells in xenogeneic, chimeric aggregates, whichcontain mouse embryonic stem cells, can differentiate into all threegerm layers and that cultured human amniotic epithelial cells expressneural and glial markers, and can synthesize and release acetylcholine,cateholamines, and dopamine. Hepatic differentiation of human amnioticepithelial cells also has been reported. Studies have reported thatcultured human amniotic epithelial cells produce albumin andα-fetroprotein and that albumin and α-fetroprotein-positivehepatocyte-like cells could be identified integrated into hepaticparenchyma following transplantation of human amniotic epithelial cellsinto the livers of severe combined inmmunodeficiency (SCID) mice. Thehepatic potential of human amniotic epithelial cells was confirmed andextended, whereby in addition to albumin and α-fetroprotein production,other hepatic functions, such as glycogen storage and expression ofliver-enriched transcription factors, such as hepatocyte nuclear factor(HNF) 3γ and HNF4α, CCAAT/enhancer-binding protein (CEBP α and β), andseveral of the drug metabolizing genes (cytochrome P450) weredemonstrated. The wide range of hepatic genes and functions identifiedin human amniotic epithelial cells has suggested that these cells may beuseful for liver-directed cell therapy (Parolini, O. et al., 2008, StemCell, 2008, 26:300-311).

Differentiation of human amniotic epithelial cells to another endodermaltissue, pancreas, also has been reported. For example, it was shown thathuman amniotic epithelial cells cultured for 2-4 weeks in the presenceof nicotinamide to induce pancreatic differentiation, expressed insulin.Subsequent transplantation of the insulin-expressing human amnioticepithelial cells corrected the hyperglycemia of streptozotocin-induceddiabetic mice. In the same setting, human amniotic mesenchymal stromalcells were ineffective, suggesting that human amniotic epithelial cells,but not human amniotic mesenchymal stromal cells, were capable ofacquiring β-cell fate (Parolini, O. et al., 2008, Stem Cell, 2008,26:300-311).

2.17.4. Mesenchymal Stromal Cells from Amnion and Chorion: hAMSC andhCMSC

Human amniotic mesenchymal cells (hAMSC) and human chorionic mesenchymalcells (hCMSC) are thought to be derived from extraembryonic mesoderm.hAMSC and hCMSC can be isolated from first-, second-, andthird-trimester mesoderm of amnion and chorion, respectively. For hAMSC,isolations are usually performed with term amnion dissected from thedeflected part of the fetal membranes to minimize the presence ofmaternal cells. For example, homogenous hAMSC populations can beobtained by a two-step procedure, whereby: minced amnion tissue istreated with trypsin to remove hAEC and the remaining mesenchymal cellsare then released by digestion (e.g., with collagenase or collagenaseand DNase). The yield from term amnion is about 1 million hAMSC and10-fold more hAEC per gram of tissue (Casey, M. and MacDonald P., BiolReprod, 1996, 55: 1253-1260).

hCMSCs are isolated from both first- and third-trimaster chorion aftermechanical and enzymatic removal of the trophoblastic layer withdispase. Chorionic mesodermal tissue is then digested (e.g., withcollagenase or collagenase plus DNase). Mesenchymal cells also have beenisolated from chorionic fetal villi through explant culture, althoughmaternal contamination is more likely (Zhang, X., et al., BiochemBiophys Res Commun, 2006, 340: 944-952; Soncini, M. et al., J Tissue EngRegen Med, 2007, 1:296-305; Zhang et al., Biochem Biophys Res Commun,2006, 351: 853-859).

The surface marker profile of cultured hAMSC and hCMSC, and mesenchymalstromal cells (MSC) from adult bone marrow are similar. All expresstypical mesenchymal markers (CD90, CD73, CD105) but are negative forhematopoietic (CD34 and CD45) and monocytic markers (CD14). Surfaceexpression of SSEA-3 and SSEA-4 and RNA for OCT-4 has been reported (WeiJ. et al., Cell Transplant, 2003, 12: 545-552; Wolbank, S. et al.,Tissue Eng, 2007, 13: 1173-1183; Alviano, F. et al., BMC Dev Biol, 2007,7: 11; Zhao, P. et al, Transplantation, 2005, 79: 528-535). Both first-and third trimester hAMSC and hCMSC express low levels of HLA-A, B, Cbut not HLA-DR, indicating an immunoprivileged status (Portmann-Lanz, C.et al, Am J Obstet Gynecol, 2006, 194: 664-673; Wolbank, S. et al.,Tissue Eng, 2007, 13: 1173-1183). The specific surface antigenexpression at passages 2-4 for amniotic mesenchymal stromal cells andhuman chorionic mesenchymal stromal cells is as follows: Positive(≥95%): CD90, CD73, CD105; Negative (≤2%): CD45, CD34, HLA-DR.

Both hAMSCs and hCMSCs differentiate toward “classic” mesodermallineages (osteogenic, chondrogenic, and adipogenic) and differentiationof hAMSC to all three germ layers-ectoderm (neural), mesoderm (skeletalmuscle, cardiomyocytic and endothelial), and endoderm (pancreatic) wasreported (Int′Anker, P. et al., Stem Cells, 2004, 22: 1338-1345;Portmann-Lanz, C. et al, Am J Obstet Gynecol, 2006, 194: 664-673;Wolbank, S. et al., Tissue Eng, 2007, 13: 1173-1183; Soncini, M. et al.,J Tissue Eng Regen Med, 2007, 1:296-305; Alviano, F., BMC Dev Biol,2007, 7: 11).

Human amniotic and chorionic cells successfully and persistently engraftin multiple organs and tissues in vivo. Human chimerism detection inbrain, lung, bone marrow, thymus, spleen, kidney, and liver after eitherintraperitoneal or intravenous transplantation of human amnion andchorion cells into neonatal swine and rats was indeed indicative of anactive migration consistent with the expression of adhesion andmigration molecules (L-selectin, VLA-5, CD29, and P-selectin ligand 1),as well as cellular matrix proteinase (MMP-2 and MMP-9) (Bailo, M. etal., Transplantation, 2004, 78:1439-1448).

2.18. Umbilical Cord

Two types of umbilical stem cells can be found, namely hematopoieticstem cells (UC-HS) and mesenchymal stem cells, which in turn can befound in umbilical cord blood (UC-MS) or in Wharton's jelly (UC-MM). Theblood of the umbilical cord has long been in the focus of attention ofresearchers as an important source of stem cells for transplantation,for several reasons: (1) it contains a higher number of primitivehematopoietic stem cells (HSC) per volume unit, which proliferate morerapidly, than bone marrow; (2) there is a lower risk of rejection aftertransplantation; (3) transplantation does not require a perfect HLAantigen match (unlike in the case of bone marrow); (4) UC blood hasalready been successfully used in the treatment of inborn metabolicerrors; and (5) there is no need for a new technology for collection andstorage of the mononuclear cells from UC blood, since such methods arelong established.

Umbilical cord (UC) vessels and the surrounding mesenchyma (includingthe connective tissue known as Wharton's jelly) derive from theembryonic and/or extraembryonic mesodermis. Thus, these tissues, as wellas the primitive germ cells, are differentiated from the proximalepiblast, at the time of formation of the primitive line of the embryo,containing MSC and even some cells with pluripotent potential. The UCmatrix material is speculated to be derived from a primitive mesenchyma,which is in a transition state towards the adult bone marrow mesenchyma(Mihu, C. et al., 2008, Romanian Journal of Morphology and Embryology,2008, 49(4):441-446).

The blood from the placenta and the umbilical cord is relatively easy tocollect in usual blood donation bags, which contain anticoagulantsubstances. Mononuclear cells are separated by centrifugation on Ficollgradient, from which the two stem cell populations will be separated:(1) hematopoietic stem cells (HSC), which express certain characteristicmarkers (CD34, CD133); and (2) mesenchymal stem cells (MSC) that adhereto the culture surface under certain conditions (e.g., modified McCoymedium and lining of vessels with Fetal Bovine Serum (FBS) or Fetal CalfSerum (FCS)). (Munn, D. et al., Science, 1998, 281: 1191-1193; Munn, D.et al., J Exp Med, 1999, 189: 1363-1372). Umbilical cord blood MSCs(UC-MS) can produce cytokines, which facilitate grafting in the donorand in vitro HSC survival compared to bone marrow MSC. (Zhang, X et al.,Biochem Biophys Res Commun, 2006, 351: 853-859).

MSCs from the umbilical cord matrix (UC-MM) are obtained by differentculture methods depending on the source of cells, e.g., MSCs from theconnective matrix, from subendothelial cells from the umbilical vein oreven from whole umbilical cord explant. They are generally well culturedin DMEM medium, supplemented with various nutritional and growthfactors; in certain cases prior treatment of vessels with hyaluronicacid has proved beneficial (Baban, B. et al., J Reprod Immunol, 2004,61: 67-77).

Exemplary factors secreted by umbilical cord tissue cells are disclosedin Zhang, X et al., Biochem Biophys Res Commun, 2006, 351: 853-859, theentire contents of which are incorporated herein by reference.

2.19. Lung Tissue

The lungs, which are paired organs that fill up the thoracic cavity,constitute an efficient air-blood gaseous exchange mechanism,accomplished by the passage of air from the mouth or nose, sequentiallythrough an oropharynx, nasopharynx, a larynx, a trachea and finallythrough a progressively subdividing system of bronchi and bronchiolesuntil it finally reaches alveoli where the air-blood gaseous exchangetakes place. A resident niche with characteristic multipotent stem cellswith c-kit positive surface profiles recently has been identifiedlocalized in small bronchioles alveoli. These stem cells express thetranscription factors, Nanog, Oct3/4, Sox2 and Klf4, that governpluripotency in embryonic stem cells. (Kajstura, J. et al., 2011, NewEngl. J. Med., 364(19):1795-1806)).

2.20. Mammary Tissue

The mammary gland is a hormone sensitive bilayered epithelial organcomprising an inner luminal epithelial layer and an outer myoepitheliallayer surrounded by a basement membrane in a stromal fat pad. Mammarystem cells with myoepithelial potential have been identified in theirniches in the terminal ducts of mammary gland. (LaBarge, 2007, Stem CellRev., 3(2): 137-146).

2.21. Dermal Tissue

Dermal tissue, such as the skin functions as the primary barrierimparting protection from environmental insults. Skin is composed of anouter epidermis and inner dermis separated by a basement membrane (BM),rich in ECM and growth factors. The BM of the epidermal-dermal junctionis composed of collagens (e.g., type IV and XVII), laminins, nidogen,fibronectin and proteoglycans that provide storage sites for growthfactors and nutrients supporting the proliferation and adhesion ofepidermal keratinocytes.

The epidermis is a solid epithelial tissue comprising keratinocytes thatare linked to each other via cellular junctions, such as desmosomes.Keratinocytes are organized into distinct layers, comprising the stratumcorneum, stratum granulosum, stratum spinosum and stratum basale. Theepidermal matrix is made up of hyaluronan and other proteoglycans,including but not limited to, desmosealin, glycipans, versican,perlecan, and syndecans. (Sandjeu and Haftek, 2009, J. Physiol.Pharmacol. 60 (S4): 23-30). Epidermal desmosomes are multimericcomplexes of transmembrane glycoprotein and cytosolic proteins with thekeratin cytoskeleton. Desmosal proteins of the epidermis predominantlybelong to the cadherin, Armadillo and plakin superfamilies.

The underlying dermis is connective tissue comprised primarily offibroblasts with occasional inflammatory cells. Embedded within thedermis are also epidermal appendages, such as hair follicles andsebaceous glands, as well as nerves and cutaneous vasculature. Thedermal ECM is essentially made of type I, III and V collagens andelastin together with noncollagenous components such as glycoproteins,proteoglycans, GAGs, cytokines and growth factors. Dermal collagens helpmediate fibroblast-matrix interactions through a number of cell surfacereceptors and proteoglycans, such as β1-integrins. (Hodde and Johnson,2007, Am. J. Clin. Dermatol. 8(2): 61-66).

During embryonic development, the epidermis originates from theectoderm, while the dermis differentiates from the mesoderm. Followinggastrulation, as mesenchymal stem cells of mesodermal origin populatethe skin, they send signals to the single epidermal layer for initiationof epidermal stratification and direct the positioning of outgrowths ofepidermal appendages, such as the hair follicles and sebaceous glands.Along with the mesenchyme, the basal layer of the epidermis organizesinto a basement membrane that is rich in ECM proteins and growthfactors. A number of different signaling pathways have been implicatedin skin morphogenesis, including but not limited to Notch, Wnt, mitogenactivated protein kinase (MAPK), nuclear factor-κB (NF-κB),transcriptional regulator, p63, the AP2 family of transcription factors,CCAAT/enhancer binding protein (C/EBP) transcriptional regulators,interferon regulatory 6 (URF6), grainyhead-like 3 (GRHL3) andKruppel-like factor (KLF4). (Blanpain and Fuchs, 2009, Nat. Rev. Mol.Cell. Biol., 10(3): 207-217).

Adult skin undergoes constant cellular turnover whereby dead skin cellsare shed and new cells are regenerated and replaced, by a process knownas skin homeostasis. Several stem cell niches with distinct surfacemarker profiles and differentiation potentials have been identified.These include, but are not limited to, epidermal stem cells ofinterfollicular epidermis; bulge stem cells and epithelial stem cells ofthe hair follicle, dermal stem cells (e.g., multipotent dermal cells,skin-derived progenitor cells, dermis-derived multipotent stem cells andfibrocytes), dermal papilla stem cells, and sebaceous gland stem cells.Collectively, these skin stem cell niches partake in maintaining skinhomeostasis with the help of growth factors and cytokines. (Zouboulis etal., 2008, Exp. Gerontol. 43: 986-997; Blanpain, 2010, Nature, 464:686-687).

Exemplary factors secreted by skin tissue cells are disclosed inBlanpain and Fuchs, 2009, Nat. Rev. Mol. Cell. Biol., 10(3): 207-217,the entire contents of which are incorporated herein by reference.

2.22. Muscular Tissue

The muscular tissue compartments are comprised of contractile muscletissue. These can be of three kinds: skeletal muscle associated with theskeletal system; cardiac muscle associated with the heart; and smoothmuscle associated with the vasculature and gastrointestinal tract.Skeletal muscle tissue fibers are striated and are voluntary infunction. Cardiac muscle fibers have characteristic intercalated discsand are involuntary in function. Smooth muscle tissue is comprised ofspindle shaped cells and is involuntary in function.

Skeletal muscles are composed of a population of quiescent myogenicprecursor cells known as satellite cells with muscle regenerating andself-renewal properties, as well as a population of multipotentmuscle-derived stem cells (MDSC) with multilineage differentiationpotential, such as mesodermal lineages including, but not limited to,myogenic lineages, adipogenic lineages, osteogenic lineages,chondrogenic lineages, endothelial and hematopoetic lineages, andectodermal lineages, including not limited to neuron-like cells. (Xu etal., 2010, Cell Tissue Res., 340: 549-567).

Skeletal muscle satellite cells are quiescent mononucleated cells thatare resident in the muscle fiber membrane, beneath the basal laminaforming distinct stem cell niches. Similar to other stem cell niches,the skeletal muscle satellite cell niche is a dynamic structure, capableof altering between inactive (quiescent) and activated states inresponse to external signals. Once activated, satellite cells have thepotential to proliferate, expand and differentiate along the myogeniclineage. The basal lamina, which serves to separate individual skeletalmuscle fibers, known as myofibers, and their associated satellite celland stem cell niches, from the cells of the interstitium, is rich incollagen type IV, perlecan, laminin, entactin, fibronectin and severalother glycoproteins and proteoglycans, that may function as receptors togrowth factors effectuating their activation by extracellular processingand modifications. In addition to these interactions provided by theECM, neighboring cells, such as endothelial cells and multipotent stemcells derived from blood vessels, such as pericytes and mesoangioblasts,or neural components, all have the potential of affecting the nichemicroenvironment. (Gopinath et al., 2008, Aging Cell, 7: 590-598).

Endogenous cardiac stem cells have also been identified in cardiac stemcell niches. (Mazhari and Hare, 2007, Nat. Clin. Pract. Cardiovasc.Med., 4(S1): S21-S26).

Vascular smooth muscle cells are derived from embryonic cardiac neuralcrest stem cells, as well as proepicardial cells and endothelialprogenitor cells. Smooth muscle differentiation is dependent on acombination of factors, including but not limited to Pax3, Tbx1, FoxC1and serum response factor, interacting with microenvironment componentsof the ECM, such as BMPs, Wnts, endothelin (ET)-1, and FGF8. In theadult, vascular smooth muscle cells undergo constant degeneration,repair and regeneration by the action of both multipotent bone-derivedmesenchymal cells as well as smooth muscle stem cells resident withinvascular smooth muscle tissue. (Hirschi and Majesky, 2004, TheAnatomical Record, Part A, 276A: 22-33).

2.23. Neural Tissue

The neural tissue compartments are comprised of neurons and theneuroglia, embedded with the neural matrix. Neural tissue is ectodermalin origin, derived from the embryonic neural plate. Neural tissue isprimarily located within the brain, spinal cord and nerves.

Resident neural stem cell niches have been identified in the adultmammalian brain, restricted to the subventricular zone as well as to thelateral ventricle and dentate gyrus subgranular zone of the hippocampus.Astrocytes, which are star-shaped nerve cells, serve as both neural stemcells as well as supporting niche cells secreting essential growthfactors that provide support for neurogenesis and vasculogenesis. Thebasal lamina and associated vasculogenesis are essential components ofthe niche. Embryonic molecular factors and signals persist within theneural stem cell niches and play critical role in neurogenesis. Neuralstem cells have VEGFR2, doublecortin and Lex (CD15) markers. Majorsignaling pathways implicated in neurogenesis include but are notlimited to Notch, Eph/ephrins, Shh, and BMPs. (Alvarez-Buylla and Lim,2004, Neuron, 41: 683-686).

Exemplary factors secreted by nerve tissue cells are listed inAlvarez-Buylla and Lim (2004), Neuron, 41: 683-686, the entire contentsof which are incorporated herein by reference.

2.24. Peritoneum

The peritoneum is the serous membrane that forms the lining of theabdominal cavity or coelom in amniotes and some invertebrates, such asannelids. It covers most of the intra-abdominal (or coelomic) organs,and is composed of a layer of mesothelium supported by a thin layer ofconnective tissue. The peritoneum supports the abdominal organs andserves as a conduit for their blood vessels, lymph vessels, and nerves.The peritoneum develops ultimately from the mesoderm of the trilaminarembryo. As the mesoderm differentiates, one region known as the lateralplate mesoderm splits to form two layers separated by an intraembryoniccoelom. These two layers develop later into the visceral and parietallayers found in all serous cavities, including the peritoneum.

3. Stem Cells

In some embodiments, the viable cells for priming can include stemcells. The term “stem cells” as used herein refers to undifferentiatedcells having high proliferative potential with the ability to self-renewthat can generate daughter cells that can undergo terminaldifferentiation into more than one distinct cell phenotype. Stem cellsare distinguished from other cell types by two characteristics. First,they are unspecialized cells capable of renewing themselves through celldivision, sometimes after long periods of inactivity. Second, undercertain physiologic or experimental conditions, they can be induced tobecome tissue- or organ-specific cells with special functions. In someorgans, such as the gut and bone marrow, stem cells regularly divide torepair and replace worn out or damaged tissues. In other organs,however, such as the pancreas and the heart, stem cells only divideunder special conditions.

Embryonic stem cells (EmSC) are stem cells derived from an embryo thatare pluripotent, i.e., they are able to differentiate in vitro intoendodermal, mesodermal and ectodermal cell types.

Adult (somatic) stem cells are undifferentiated cells found amongdifferentiated cells in a tissue or organ. Their primary role in vivo isto maintain and repair the tissue in which they are found. Adult stemcells have been identified in many organs and tissues, including brain,bone marrow, peripheral blood, blood vessels, skeletal muscles, skin,teeth, gastrointestinal tract, liver, ovarian epithelium, and testis.Adult stem cells are thought to reside in a specific area of eachtissue, known as a stem cell niche, where they may remain quiescent(non-dividing) for long periods of time until they are activated by anormal need for more cells to maintain tissue, or by disease or tissueinjury. Examples of adult stem cells include, but not limited to,hematopoietic stem cells, mesenchymal stem cells, neural stem cells,epithelial stem cells, and skin stem cells.

3.1. Hematopoietic Stem Cells (HSCs)

Hematopoietic stem cells (also known as the colony-forming unit of themyeloid and lymphoid cells (CFU-M,L), or CD34+ cells) are rarepluripotential cells within the blood-forming organs that areresponsible for the continued production of blood cells during life.While there is no single cell surface marker exclusively expressed byhematopoietic stem cells, it generally has been accepted that human HSCshave the following antigenic profile: CD 34+, CD59+, Thy1+(CD90),CD38low/−, C-kit−/low and, lin−. CD45 is also a common marker of HSCs,except platelets and red blood cells. HSCs can generate a variety ofcell types, including erythrocytes, neutrophils, basophils, eosinophils,platelets, mast cells, monocytes, tissue macrophages, osteoclasts, andthe T and B lymphocytes. The regulation of hematopoietic stem cells is acomplex process involving self-renewal, survival and proliferation,lineage commitment and differentiation and is coordinated by diversemechanisms including intrinsic cellular programming and externalstimuli, such as adhesive interactions with the micro-environmentalstroma and the actions of cytokines.

Different paracrine factors are important in causing hematopoietic stemcells to differentiate along particular pathways. Paracrine factorsinvolved in blood cell and lymphocyte formation are called cytokines.Cytokines can be made by several cell types, but they are collected andconcentrated by the extracellular matrix of the stromal (mesenchymal)cells at the sites of hematopoiesis. For example, granulocyte-macrophagecolony-stimulating factor (GM-CSF) and the multilineage growth factorIL-3 both bind to the heparan sulfate glycosaminoglycan of the bonemarrow stroma. The extracellular matrix then presents these factors tothe stem cells in concentrations high enough to bind to their receptors.

3.2. Mesenchymal Stem Cells (MSCs)

Mesenchymal stem cells (MSCs) (also known as bone marrow stromal stemcells or skeletal stem cells) are non-blood adult stem cells found in avariety of tissues. They are characterized by their spindle-shapemorphologically; by the expression of specific markers on their cellsurface; and by their ability, under appropriate conditions, todifferentiates along a minimum of three lineages (osteogenic,chondrogenic, and adipogenic).

No single marker that definitely delineates MSCs in vivo has beenidentified due to the lack of consensus regarding the MSC phenotype, butit generally is considered that MSCs are positive for cell surfacemarkers CD105, CD166, CD90, and CD44 and that MSCs are negative fortypical hematopoietic antigens, such as CD45, CD34, and CD14. Other MSCmarkers can include CD271, CD73, CD 29, CD117, CD200, CD348, and Stro-1.As for the differentiation potential of MSCs, studies have reported thatpopulations of bone marrow-derived MSCs have the capacity to developinto terminally differentiated mesenchymal phenotypes both in vitro andin vivo, including bone, cartilage, tendon, muscle, adipose tissue, andhematopoietic-supporting stroma. Studies using transgenic and knockoutmice and human musculoskeletal disorders have reported that MSCdifferentiate into multiple lineages during embryonic development andadult homeostasis.

Analyses of the in vitro differentiation of MSCs under appropriateconditions that recapitulate the in vivo process have led to theidentification of various factors essential for stem cell commitment.Among them, secreted molecules and their receptors (e.g., transforminggrowth factor-β), extracellular matrix molecules (e.g., collagens andproteoglycans), the actin cytoskeleton, and intracellular transcriptionfactors (e.g., Cbfal/Runx2, PPARγ, Sox9, and MEF2) have been shown toplay important roles in driving the commitment of multipotent MSCs intospecific lineages, and maintaining their differentiated phenotypes.

For example, it has been shown that osteogenesis of MSCs, both in vitroand in vivo, involves multiple steps and the expression of variousregulatory factors. During osteogenesis, multipotent MSCs undergoasymmetric division and generate osteoprecursors, which then progress toform osteoprogenitors, preosteoblasts, functional osteoblasts, andeventually osteocytes. This progression from one differentiation stageto the next is accompanied by the activation and subsequent inactivationof transcription factors, i.e., Cbfal/Runx2, Msx2, Dlx5, Osx, andexpression of bone-related marker genes, i.e., osteopontin, collagentype I, alkaline phosphatase, bone sialoprotein, and osteocalcin.

Members of the Wnt family also have been shown to impact MSCosteogenesis. Wnts are a family of secreted cysteine-rich glycoproteinsthat have been implicated in the regulation of stem cell maintenance,proliferation, and differentiation during embryonic development.Canonical Wnt signaling increases the stability of cytoplasmic β-cateninby receptor-mediated inactivation of GSK-3 kinase activity and promotesβ-catenin translocation into the nucleus. The active β-catenin/TCF/LEFcomplex then regulates the transcription of genes involved in cellproliferation. In humans, mutations in the Wnt co-receptor, LRP5, leadto defective bone formation. “Gain of function” mutation results in highbone mass, whereas “loss of function” causes an overall loss of bonemass and strength, indicating that Wnt signaling is positively involvedin embryonic osteogenesis. Canonical Wnt signaling pathway alsofunctions as a stem cell mitogen via stabilization of intracellularβ-catenin and activation of the β-catenin/TCF/LEF transcription complex,resulting in activated expression of cell cycle regulatory genes, suchas Myc, cyclin D1, and Msx1. When MSCs are exposed to Wnt3a, aprototypic canonical Wnt signal, under standard growth mediumconditions, they show markedly increased cell proliferation and adecrease in apoptosis, consistent with the mitogenic role of Wnts inhematopoietic stem cells. However, exposure of MSCs to Wnt3a conditionedmedium or overexpression of ectopic Wnt3a during osteogenicdifferentiation inhibits osteogenesis in vitro through β-cateninmediated down-regulation of TCF activity. The expression of severalosteoblast specific genes, e.g., alkaline phosphatase, bonesialoprotein, and osteocalcin, is dramatically reduced, while theexpression of Cbfal/Runx2, an early osteo-inductive transcription factoris not altered, implying that Wnt3a-mediated canonical signaling pathwayis necessary, but not sufficient, to completely block MSC osteogenesis.On the other hand, Wnt5a, a typical non-canonical Wnt member, has beenshown to promote osteogenesis in vitro. Since Wnt3a promotes MSCproliferation during early osteogenesis, it is thought likely thatcanonical Wnt signaling functions in the initiation of early osteogeniccommitment by increasing the number of osteoprecursors in the stem cellcompartment, while non-canonical Wnt drives the progression ofosteoprecursors to mature functional osteoblasts.

3.3. Epithelial Stem Cells

An epithelial membrane is a continuous multicellular sheet composed ofan epithelium adhered to underlying connective tissue. Epithelialmembranes can be cutaneous (e.g. skin), mucous (e.g., gastrointestinallining) and serous (e.g. pleural lining, pericardial lining andperitoneal lining).

Epithelial stem cells line the gastrointestinal tract in deep crypts andgive rise to absorptive cells, goblet cells, paneth cells, andenteroendocrine cells.

3.4. Neural Stem Cells

The adult mammalian brain contains multipotent neural stem cells (NSCs)that have the capacity to self-renew and are responsible forneurogenesis and maintenance of specific regions of the adult brain.Neural stem cells can generate astrocytes, oligodendrocytes, andneurons. Self-renewal and differentiation of neural stem cells aredirected by interactions within a complex network of intrinsicregulators and extrinsic factors. Recent proteomic analyses haveidentified a horde of transcription factors belonging to theWnt/β-catenin, Notch and Sonic Hedgehog (shh) pathways, in addition toepigenetic modifications, microRNA networks and extrinsic growth factornetworks, including but not limited to the FGFs and BMPs. (Yun ey al.,2010, J. Cell. Physiol. 225: 337-347).

With the advent of high throughput microarray and proteomictechnologies, a number of different molecular signatures of neural stemcells have been identified, including but not limited to CD133/promini,nestin, NCAM, the HMG-box transcription factor, Sox2 and the bHLHprotein, Olig2. (Holmberg et al., 2011, PLoS One., 6(3): e18454;Hombach-Klonisch et al., 2008, J. Mol. Med. 86(12): 1301-1314).

3.5. Skin Stem Cells

Several different adult stem cell populations with distinct molecularsignatures are responsible for maintaining skin homeostasis. Theseinclude, but are not limited to, epidermal stem cells of theinterfollicular region, epidermal stem cells of the hair follicle (alsoknown as the bulge stem cells), dermal stem cells, dermal papilla stemcells, and sebaceous gland stems. The epidermal stem cells areectodermal in origin while the dermal stem cells originate from themesoderm and are mesenchymal in nature. (Zouboulis et al., 2008, Exp.Gerontol., 43: 986-997).

The interfollicular epidermal stem cells reside in the basal layer ofthe epidermis and give rise to keratinocytes, which migrate to thesurface of the skin and form a protective layer. A diverse range ofmolecular signatures has been described for such epidermal stem cellsincluding but not limited to high α6-integrin, low CD71, high Delta 1(Notch signaling ligand) and high CD200 expression levels. Thefollicular stem cells located at the base of hair follicles give rise toboth hair follicle and to the epidermis. These are characterized byCytokeratin 15 (K15) immunostaining and high levels of β1-integrin.Dermal stem cell marker proteins include but are not limited to nestin,fibronectin and vimentin, the surface markers for dermal papilla stemcells include mesenchymal stem cell markers such as for example CD44,CD73 and CD90 and sebaceous stem cells express keratin 14. (Zouboulis etal., 2008, Exp. Gerontol., 43: 986-997).

In addition, adult somatic cells can be reprogrammed to enter anembryonic stem cell-like state by being forced to express a set oftranscription factors, for example, Oct-3/4 (or Pou5f1, the Octamertranscription factor-3/4), the Sox family of transcription factors(e.g., Sox-1, Sox-2, Sox-3, and Sox-15), the Klf family transcriptionfactors (Klf-1, Klf-2, Klf-4, and Klf-5), and the Myc family oftranscription factors (e.g., c-Myc, N-Myc, and L-Myc). For example,human inducible Pluripotent Stem cells (iPSCs) are cells reprogrammed toexpress transcription factors that express stem cell markers and arecapable of generating cells characteristic of all three germ layers(i.e., ectoderm, mesoderm, and endoderm).

3.6. Stem Cell Niches

Adult tissue compartments contain endogenous niches of adult stem cellsthat are capable of differentiating into diverse cell lineages ofdetermined endodermal, mesodermal or ectodermal fate depending on theirlocation in the body. For example, in the presence of an appropriate setof internal and external signals, bone marrow-derived adulthematopoietic stem cells (HSCs) have the potential to differentiate intoblood, endothelial, hepatic and muscle cells; brain-derived neural stemcells (NSCs) have the potential to differentiate into neurons,astrocytes, oligodendrocytes and blood cells; gut- and epidermis-derivedadult epithelial stem cells (EpSCs) have the potential to give rise tocells of the epithelial crypts and epidermal layers; adipose-derivedstem cells (ASCs) have the potential to give rise to fat, muscle,cartilage, endothelial cells, neuron-like cells and osteoblasts; andbone-marrow-derived adult mesenchymal stem cells (MSCs) have thepotential to give rise to bone, cartilage, tendon, adipose, muscle,marrow stroma and neural cells.

Endogenous adult stem cells are embedded within the ECM component of agiven tissue compartment, which, along with support cells, form thecellular niche. Such cellular niches within the ECM scaffold togetherwith the surrounding microenvironment contribute important biochemicaland physical signals, including growth factors and transcription factorsrequired to initiate stem cell differentiation into committed precursorscells and subsequent precursor cell maturation to form adult tissuecells with specialized phenotypic and functional characteristics.

4. Extracellular Matrix

The viable cells of the present invention can be endogenous to andresident within the host tissue compartment, e.g., within the ECM.During priming and after priming (e.g., when grafted), the cells cansecrete extracellular macromolecules or other factors into the ECM, aswell as migrate in the ECM. Where cell-free grafts are needed, the cellscan be removed after priming, leaving behind ECM. Alternatively, wherenon-viable grafts are required, the cells are devitalized but notremoved from the graft. Thus, ECM plays an important role in someembodiments of the present invention.

The ECM is an intricate network of secreted extracellular macromoleculesthat largely fills the extracellular space in the tissue compartmentsand comprises large polymeric complexes of glycosaminoglycans (GAGs) andproteoglycans. GAGs are negatively charged unbranched polysaccharidechains comprising repeating disaccharide units. Each repeatingdisaccharide unit of a GAG chain contains an amino sugar(N-acetylglucosamine or N-acetyl glucosamine), which in most cases issulfated, and an -uronic acid (glucuronic or iduronic acid). Four maintypes of GAG molecules are distinguished based on sugar residues, typeof linkage, number and location of sulfate groups: (1) hyaluronan; (2)chondroitan sulfate and dermatan sulfate; (3) heparan sulfate andheparin; and (4) keratin sulfate.

GAG chains are inflexible and tend to adopt extended conformationsoccupying a huge volume relative to their mass, forming gels even at lowconcentrations. Their high density of negative charges attracts cations,such as Na+, that are effective in osmotic absorption of large amountsof water into the matrix. This creates high turgor enabling the ECM towithstand compressive forces.

Hyaluronan (also termed hyaluronic acid or hyaluronate) (HA), whichcomprises a regular repeating sequence of up to 25,000 nonsulfateddisaccharide units, serves many functions, many of which depend on thebinding of HA-binding proteins and proteoglycans, which are eitherthemselves constituents of the ECM or are integral constituents of cellsurfaces. For example, HA resists compressive forces in joints as amajor constituent of joint fluid serving as a lubricant; serves as aspace filler during embryonic development; creates a cell-free space inepithelial compartment to allow cell migration during the formation ofheart, cornea and other organs; and plays a role in wound repair. ExcessHA is usually degraded by hyaluronidase.

All GAGs, except for HA, are covalently linked to proteins in the formof proteoglycans. During their synthesis, the polypeptide chain ofproteoglycans is synthesized on membrane-bound ribosomes and threadedinto the lumen of endoplasmic reticulum, from which they are sorted inthe Golgi apparatus, and assembled with polysaccharide chains. Whilestill in the Golgi, proteoglycans undergo a series of sequential andcoordinated sulfation and epimerization reactions to produce sulfatedproteoglycans. Sulfated and nonsulfated proteoglycans then travelthrough the Golgi network and are ultimately secreted into the ECM byexocytosis with the help of secretory vesicles.

Proteoglycans are heterogenous molecules, with core proteins ranging inmolecular weight from 10 kD to about 600 kD and with attached GAG chainsvarying in number and type, further modified by a complex variablepattern of sulfate groups. At least one of the proteoglycan sugar sidechains is a GAG; the core protein is usually a glycoprotein, but maycomprise up to 95% carbohydrate by weight, mostly as long unbranched GAGchains up to at least 80 sugar residues long.

Proteoglycans along with their attached GAG chains regulate theactivities of secreted macromolecules. They can serve as selectivemolecular sieves regulating a size-based trafficking of molecules andcells, and play a role in cell-cell signaling. Proteoglycans modulatethe activities of secreted factors, such as growth factors andcytokines, by binding to them For example, binding of fibroblast growthfactor (FGF) to heparan sulfate chains of proteoglycans is required forFGF activation of its cell surface receptors. On the other hand, forexample, binding of a ubiquitous growth regulatory factor, such astransforming growth factor β (TGF-β) to core proteins of several ECMproteoglycans, such as decorin, results in inhibition of TGF-β activity.Proteoglycans also bind and regulate the activities of other types ofsecreted proteins, such as proteases and protease inhibitors.Cell-surface proteoglycans also may act as co-receptors: for example,syndecan binds to FGF and presents it to the FGF-receptor. Similarly,betaglycan binds to TGF-β and presents it to TGF-β receptors.

Collagens and elastin are the major fibrous proteins of the ECM.Collagens comprise a family of highly characteristic fibrous proteinsand are a major component of skin and bone. Collagen fibers consist ofglobular units of the collagen subunit tropocollagen. Each tropocollagensubunit molecule comprises three polypeptide chains, called a chains,each exhibiting a left-handed helical conformation, that are wrappedaround each other in a right-handed coiled coil structure, also called atriple helix or super helix. A characteristic feature of collagen is arepeating tripeptide unit comprising Glycine-Proline-X orGlycine-X-Hydroxyproline, where X may be any amino acid. The presence ofGlycine at every third position in a collagen unit is critical formaintaining the coiled coil structure, since each repeating glycineresidue sits on the interior axis of the helix, which sterically hindersbulkier sidechains. Prolines and hydroxyprolines help stabilize thetriple helix. Collagen is secreted as procollagen molecules, whichundergo proteolytic processing and subsequent assembly to formcollagenous fibrils. Collagens are highly glycosylated during proteintrafficking through intracellular secretory pathways.

Collagens are classified into various types depending on the nature oftheir a chains. Collagen molecules composition, class and distributionare reviewed extensively by Shoulders and Raines, Annu. Rev. Biochem.2009, 78: 929-958 and Kelly et al. 1984, Bailey's Textbook ofMicroscopic Anatomy, Williams and Wilkins, 18th edition.

A network of elastic fibers in the ECM offers resilience and elasticityso that organs are able to recoil following transient stretch. Elasticfibers primarily comprise the fibrous protein elastin, a highlyhydrophobic protein about 750 amino acids in length that is rich inproline and glycine, is not glycosylated and is low in hydroxyprolineand hyroxylysine. Elastin molecules are secreted into the ECM andassemble into elastic fibers close to the plasma membrane. Uponsecretion, elastin molecules become highly cross-linked to form anextensive network of fibers and sheets.

The ECM also comprises many non-collagen adhesive proteins, usually withmultiple domains containing binding sites of other macromolecules andfor cell-surface receptors. One such adhesive protein, fibronectin, is alarge glycoprotein comprising two subunits joined by a pair of disulfidebonds near the carboxy termini. Each subunit is folded into a series ofrod-like domains interspersed by regions of flexible polypeptide chains.Each domain further comprises repeating modules of various types. Onemajor type of fibronectin repeating module, called type III fibronectinrepeat, is about 90 amino acids in length and occurs at least 15 timesin each subunit. Fibronectin type III repeats have characteristicArg-Gly-Asp (RGD) tripeptide repeats that function as binding sites forother proteins such as collagen, heparin or cell surface receptors.Fibronectin not only plays an important role in cell adhesion to theECM, but also in guiding cell migration in vertebrate embryos.

Laminin, another adhesive glycoprotein of the ECM, is a majorconstituent (along with type IV collagen and another glycoprotein,entactin) of the basal lamina, a tough sheet of ECM formed at the baseof epithelial cells. Laminin is a large flexible complex, about 850 kDin molecular weight, with three very long polypeptide chains arranged inthe form of an asymmetric cross held together with disulfide bonds.Laminin contains numerous functional domains, e.g., one binds to type IVcollagen, one to heparan sulfate, one to entactin and two or more tolaminin receptor proteins on the cell surface.

5. Growth Factors

Growth factors can, in some embodiments, be used to prime the viablecells. Growth factors are extracellular polypeptide molecules that bindto a cell-surface receptor triggering an intracellular signalingpathway, leading to proliferation, differentiation, or other cellularresponse. These pathways stimulate the accumulation of proteins andother macromolecules, and they do so by both increasing their rate ofsynthesis and decreasing their rate of degradation. One intracellularsignaling pathway activated by growth factor receptors involves theenzyme PI 3-kinase, which adds a phosphate from ATP to the 3 position ofinositol phospholipids in the plasma membrane. The activation of PI3-kinase leads to the activation of several protein kinases, includingS6 kinase. The S6 kinase phosphorylates ribosomal protein S6, increasingthe ability of ribosomes to translate a subset of mRNAs, most of whichencode ribosomal components, as a result of which, protein synthesisincreases. When the gene encoding S6 kinase is inactivated inDrosophila, cell numbers are normal, but cell size is abnormally small,and the mutant flies are small. Growth factors also activate atranslation initiation factor called eIF4E, further increasing proteinsynthesis and cell growth.

Growth factor stimulation also leads to increased production of the generegulatory protein Myc, which plays a part in signaling by mitogens. Mycincreases the transcription of a number of genes that encode proteinsinvolved in cell metabolism and macromolecular synthesis. In this way,it stimulates both cell metabolism and cell growth.

Some extracellular signal proteins, including platelet-derived growthfactor (PDGF), can act as both growth factors and mitogens, stimulatingboth cell growth and cell-cycle progression. This functional overlap isachieved in part by overlaps in the intracellular signaling pathwaysthat control these two processes. The signaling protein Ras, forexample, is activated by both growth factors and mitogens. It canstimulate the PI3-kinase pathway to promote cell growth and theMAP-kinase pathway to trigger cell-cycle progression. Similarly, Mycstimulates both cell growth and cell-cycle progression. Extracellularfactors that act as both growth factors and mitogens help ensure thatcells maintain their appropriate size as they proliferate.

Since many mitogens, growth factors, and survival factors are positiveregulators of cell-cycle progression, cell growth, and cell survival,they tend to increase the size of organs and organisms. In some tissues,however, cell and tissue size also is influenced by inhibitoryextracellular signal proteins that oppose the positive regulators andthereby inhibit organ growth. The best-understood inhibitory signalproteins are TGF-β and its relatives. TGF-β inhibits the proliferationof several cell types, either by blocking cell-cycle progression in G1or by stimulating apoptosis. TGF-β binds to cell-surface receptors andinitiates an intracellular signaling pathway that leads to changes inthe activities of gene regulatory proteins called Smads. This results incomplex changes in the transcription of genes encoding regulators ofcell division and cell death.

Bone morphogenetic protein (BMP), a TGF-β family member, helps triggerthe apoptosis that removes the tissue between the developing digits inthe mouse paw. Like TGF-β, BMP stimulates changes in the transcriptionof genes that regulate cell death.

Non-limiting, exemplary growth factors are discussed in detail below.Other growth factors known in the art are also included in the presentinvention.

5.1. Fibroblast Growth Factor (FGF)

The fibroblast growth factor (FGF) family currently has over a dozenstructurally related members. FGF1 is also known as acidic FGF; FGF2 issometimes called basic FGF (bFGF); and FGF7 sometimes goes by the namekeratinocyte growth factor. Over a dozen distinct FGF genes are known invertebrates; they can generate hundreds of protein isoforms by varyingtheir RNA splicing or initiation codons in different tissues. FGFs canactivate a set of receptor tyrosine kinases called the fibroblast growthfactor receptors (FGFRs). Receptor tyrosine kinases are proteins thatextend through the cell membrane. The portion of the protein that bindsthe paracrine factor is on the extracellular side, while a dormanttyrosine kinase (i.e., a protein that can phosphorylate another proteinby splitting ATP) is on the intracellular side. When the FGF receptorbinds an FGF (and only when it binds an FGF), the dormant kinase isactivated, and phosphorylates certain proteins within the respondingcell, activating those proteins.

FGFs are associated with several developmental functions, includingangiogenesis (blood vessel formation), mesoderm formation, and axonextension. While FGFs often can substitute for one another, theirexpression patterns give them separate functions. FGF2 is especiallyimportant in angiogenesis, whereas FGF8 is involved in the developmentof the midbrain and limbs.

The expression levels of angiogenic factors, such as VEGF, IGF, PDGF,HGF, FGF, TGFm Angiopoeitin-1, and stem cell factor (SCF) have beenfound to differ amongst bone-derived-, cartilage-derived-, andadipose-derived MSCs. (Peng et al., 2008, Stems Cells and Development,17: 761-774).

5.2. Insulin-Like Growth Factor (IGF-1)

IGF-1, a hormone similar in molecular structure to insulin, hasgrowth-promoting effects on almost every cell in the body, especiallyskeletal muscle, cartilage, bone, liver, kidney, nerves, skin,hematopoietic cell, and lungs. It plays an important role in childhoodgrowth and continues to have anabolic effects in adults. IGF-1 isproduced primarily by the liver as an endocrine hormone as well as intarget tissues in a paracrine/autocrine fashion. Production isstimulated by growth hormone (GH) and can be retarded by undernutrition,growth hormone insensitivity, lack of growth hormone receptors, orfailures of the downstream signaling molecules, including SHP2 andSTAT5B. Its primary action is mediated by binding to its specificreceptor, the Insulin-like growth factor 1 receptor (IGF1R), present onmany cell types in many tissues. Binding to the IGF1R, a receptortyrosine kinase, initiates intracellular signaling; IGF-1 is one of themost potent natural activators of the AKT signaling pathway, astimulator of cell growth and proliferation, and a potent inhibitor ofprogrammed cell death. IGF-1 is a primary mediator of the effects ofgrowth hormone (GH). Growth hormone is made in the pituitary gland,released into the blood stream, and then stimulates the liver to produceIGF-1. IGF-1 then stimulates systemic body growth. In addition to itsinsulin-like effects, IGF-1 also can regulate cell growth anddevelopment, especially in nerve cells, as well as cellular DNAsynthesis.

5.3. Transforming Growth Factor Beta (TGF-β)

There are over 30 structurally related members of the TGF-β superfamily,and they regulate some of the most important interactions indevelopment. The proteins encoded by TGF-β superfamily genes areprocessed such that the carboxy-terminal region contains the maturepeptide. These peptides are dimerized into homodimers (with themselves)or heterodimers (with other TGF-β peptides) and are secreted from thecell. The TGF-β superfamily includes the TGF-β family, the activinfamily, the bone morphogenetic proteins (BMPs), the Vg-1 family, andother proteins, including glial-derived neurotrophic factor (GDNF,necessary for kidney and enteric neuron differentiation) and Müllerianinhibitory factor, which is involved in mammalian sex determination.TGF-β family members TGF-β1, 2, 3, and 5 are important in regulating theformation of the extracellular matrix between cells and for regulatingcell division (both positively and negatively). TGF-β1 increases theamount of extracellular matrix epithelial cells make both by stimulatingcollagen and fibronectin synthesis and by inhibiting matrix degradation.TGF-βs may be critical in controlling where and when epithelia canbranch to form the ducts of kidneys, lungs, and salivary glands.

The members of the BMP family were originally discovered by theirability to induce bone formation. Bone formation, however, is only oneof their many functions, and they have been found to regulate celldivision, apoptosis (programmed cell death), cell migration, anddifferentiation. BMPs can be distinguished from other members of theTGF-β superfamily by their having seven, rather than nine, conservedcysteines in the mature polypeptide. The BMPs include proteins such asNodal (responsible for left-right axis formation) and BMP4 (important inneural tube polarity, eye development, and cell death).

5.4. Neural Epidermal Growth-Factor-Like 1 (NELL1)

Neural epidermal growth-factor-like 1 (NEL-like 1, NELL1) is a gene thatencodes an 810-amino acid polypeptide, which trimerizes to form a matureprotein involved in the regulation of cell growth and differentiation.The neural epidermal growth-factor-like (nel) gene first was detected inneural tissue from an embryonic chicken cDNA library, and its humanorthologue NELL1 was discovered later in B-cells. Studies have reportedthe presence of NELL in various fetal and adult organs, including, butnot limited to, the brain, kidneys, colon, thymus, lung, and smallintestine.

Much of what is known about NELL1 concerns its role in bone development.See, e.g., U.S. Pat. Nos. 7,884,066, 7,833,968, 7,807,787, 7,776,361,7,691,607, 7,687,462, 7,544,486, and 7,052,856, the entire contents ofwhich are incorporated herein by reference. It generally is believedthat during osteogenic differentiation, NELL1 signaling may involve anintegrin-related molecule and tyrosine kinases that are triggered byNELL1 binding to a NELL1 specific receptor and a subsequent formation ofan extracellular complex. As thus far understood, in human NELL1(hNELL1), the laminin G domain comprises about 128 amino acid residuesthat show a high degree of similarity to the laminin G domain ofextracellular matrix (“ECM”) proteins, such as human laminin α3 chain(hLAMA3), mouse laminin α3 chain (mLAMA3), human collagen 11 α3 chain(hCOLA1), and human thrombospondin-1 (hTSP1). This complex facilitateseither activation of Tyr-kinases, inactivation of Tyr phosphatases, orintracellular recruitment of Tyr-phosphorylated proteins. The ligandbound integrin (cell surface receptors that interact with ECM proteinssuch as, for example, laminin 5, fibronectin, vitronectin, TSP1/2)transduces the signals through activation of the focal adhesion kinase(FAK) followed by indirect activation of the Ras-MAPK cascade, and thenleads to osteogenic differentiation through Runx2; the laminin G domainis believed to play a role in the interaction between integrins and a 67kDa laminin receptor.

The NELL1 protein is a secreted cytoplasmic heterotrimeric protein. Thecomplete role NELL1 plays in vivo remains unknown. Several studies haveindicated that NELL1 may play a role in bone formation, inflammatorybowel disease, and esophageal adenocarcinoma, among others. It generallyis believed that NELL1 induces osteogenic differentiation and boneformation of osteoblastic cells during development. Studies have shownthat the NELL1 protein (1) transiently activates the mitogen-activatedprotein kinase (“MAPK”) signaling cascade (which is involved in variouscellular activities such as gene expression, mitosis, differentiation,proliferation and apotosis); and (2) induces phosphorylation of Runx2 (atranscription factor associated with osteoblast differentiation).Consequently, it generally is believed that upon binding to a specificreceptor, NELL1 transduces an osteogenic signal through activation ofcertain Tyr-kinases associated with the Ras-MAPK cascade, whichultimately leads to osteogenic differentiation. Studies have shown thatbone development is severely disturbed in transgenic mice whereover-expression of NELL1 has been shown to lead to craniosynotosis(premature ossification of the skull and closure of the sutures) andNELL1 deficiency manifests in skeletal defects due to reducedchondrogenesis and osteogenesis.

6. Tissue and Cell Priming

Methods and compositions for priming tissues and resident cells thereinare provided. In some embodiments, the primed tissues and/or cells canbe used in grafting, surgical, medical or other therapeutic procedures.For example, grafts can contain viable cells that have been primed orpre-conditioned during processing prior to clinical usage. These viablecells are endogenous to the tissue (e.g., resident within the tissuecompartment) and are not isolated or dissociated from the tissue. Inother embodiments, grafts can contain non-viable cells or cell remnantsthat have been primed or pre-conditioned during processing prior toclinical usage. In still other embodiments, grafts can have had viablecells that have been primed or pre-conditioned and then removed duringprocessing prior to clinical usage. Grafts can also have a combinationof viable cells, non-viable cells, and/or removed cells. Priming canprovide an improved and/or more rapid therapeutic effect such asenhanced healing, compared to traditional or non-primed graft.

In some embodiments, the primed tissue or cells can be used inconnection with the tissuegenic cells disclosed in U.S. Pat. Nos.8,834,928, 8,883,210 and 9,352,003, all of which are incorporated hereinby reference in their entirety.

Without wishing to be bound by theory, it is believed that at least someof the viable cells, resident in the host tissue, can be reprogramedduring priming. For example, in response to priming stimuli, cells canbe induced to differentiate, or de-differentiate, as illustrated in FIG.1A. Further, as illustrated in FIG. 1B, stem cells can be induced toexit self-renewal cycles and differentiate into a specific cell type,de-differentiate into stem cells before re-differentiation at a latertime point, or trans-differentiate into multiple cell types. Selectingproper priming conditions can direct the cells to any of the foregoingpaths. In addition, cells can also be reprogramed to exit the apoptosiscascade, and be kept alive in conditions that would normally be fatal(e.g., in a harvested graft tissue). It should be noted that complextissues may contain many different cell types at various development anddifferentiation stages, and thus, different reprogramming processes cantake place simultaneously within the same tissue during priming. Theseat least partially reprogramed cells when grafted, can directlyparticipate in healing process and/or actively secret factors thatelicit healing.

Various tissues and endogenous cells resident therein (e.g., thosedisclosed herein) can be subjected to the priming or pre-conditioningmethods of the present invention. Non-limiting examples of tissuescontaining viable cells that can be primed include, placenta, amnion,chorion, umbilical cord, Wharton's jelly, bone, cartilage (e.g.,articular, auricular, costal), spinal disc, periosteum, adipose,meniscus, muscle, tendon, ligament, skin, cardiovascular, peritoneum,fascia, nerve, cornea, visceral organs, reproductive tissues, hairfollicles, foreskin, and dental tissue. In some embodiments, the tissuecan be an allograft, autograft or xenograft. The cells can include stemcells, non-terminally differentiated cells and/or differentiated cellsas disclosed herein.

In some embodiments, the viable cells can be primed by exposing tobiochemical stimulation with soluble factors (e.g., growth-inductivecomponent, medium component, inhibitor, antioxidant, vitamin, enzyme,adipokine, cytokine, growth factor, hormone, steroid anddifferentiation-inducing factor) for a predetermined duration of time.

Growth-inductive components can include but are not limited to mitogensand growth factors. Examples of growth-inductive components includePDGF, EGF, TGF-β, and FGF.

Antioxidants can include but are not limited to Vitamin C (ascorbicacid), Vitamin E, glutathione, lipoic acid, melatonin, uric acid,carotenes, ubiquinol, resveratrol, tocopherols, polyphenols, selenium,and flavanoids.

Medium components include but are not limited to glycine, glutamine,sodium pyruvate, and other amino acids.

Enzymes include but are not limited to collagenase, dispase,metalloproteinase, trypsin, telomerase, hyaluronidase, elastase, papain,pronase, and bromelain.

Inhibitors can include but are not limited to agents that are inhibitcell death or apoptosis, cell division, expression of specific genes,differentiation, senescence, or protein synthesis. Apoptosis inhibitorsinclude but are not limited to agents known to inhibit caspases such asthe Bcl-2 family, cytokine response modifier A (crmA) family, andinhibitors of apoptosis proteins (IAP) family.

Hormones include but are not limited to dexamethasone, Vitamin D(calcitriol), melatonin, calcitonin, epinephrine, insulin, leptin,progesterone, estrogen, and androgen.

Agents that induce expression of anti-microbial proteins include but arenot limited to phenylbutyrate, Vitamin A, Vitamin D, cholic acid,chenodeoxycholic acid, lithocholic acid, sulforaphane, and retinoicacid.

Anti-inflammatory agents include but are not limited to curcumin,capsaicin, betaine, batalaine, aspirin, ibuprofen, naproxen, resolving,protectin, maresin, omega-3 fatty acids, and TGF-β.

Angiogenic agents include but are not limited to VEGF, PDGF, bFGF,TGF-β, placental growth factor (PIGF/PGF), angiopoietin (Ang)-2,angiogenin ephrin, and plasminogen activators.

Differentiation agents or differentiation-inducing factors include butare not limited to dexamethasone, β-glycerolphosphate, IBMX,indomethacin, β-mercaptoethanol, retinoic acid, BDNF, FGF, GDNF, SHH,forskolin, BMP, glucose, and insulin.

The TIMP (Tissue Inhibitor of Metalloproteinase) family can also be usedas a stimulus. This family is related to tissue remodeling in woundhealing. These include at least TIMP1, TIMP2, TIMP3, and TIMP4.

In some embodiments, suitable adipokines can include, for example,angiopoietin-1, angiopoietin-2, VEGF, transforming growth factor beta(TGF-β), hepatic growth factor (HGF), stromal derived growth factor 1(SDF-1), TNF-α, resistin, leptin, tissue factor, placental growth factor(PGF), insulin like growth factor (IGF), and monobutyrin.

In some embodiments, suitable cytokines can include, for example, SDF-la(stromal cell-derived factor 1), Bone Morphogenic Proteins (BMPs),Epidermal Growth Factors (EGFs), Fibroblast Growth Factors (FGFs),Platelet-Derived Growth Factors (PDGFs), Insulin-like Growth Factor-1(IGF-1), Transforming Growth Factors (TGFs), Bone-Derived Growth Factors(BDGFs), Cartilage-Derived Growth Factor (CDGF), Skeletal Growth Factor(hSGF), Interleukin-1 (IL-1), and macrophage-derived factors.

In some embodiments, suitable growth factors can include, for example,BMP-2, rhBMP-2, BMP-4, rhBMP-4, BMP-6, rhBMP-6, BMP-7 [OP-1], rhBMP-7,GDF-5, Statin, LIM mineralization protein, Nel-1 protein, neuralepidermal growth-factor-like 1 (Nel-like 1, NELL1), platelet derivedgrowth factor (PDGF), vascular endothelial growth factor (VEGF),transforming growth factor β (TGF-β), insulin-related growth factor-I(IGF-I), insulin-related growth factor-II (IGF-II), fibroblast growthfactor (FGF), FGF-2, FGF-5, beta-2-microglobulin (BDGF II), and rhGDF-5.

In some embodiments, suitable hormones and steroids can include, forexample, aldosterone, androstenedione, calcidiol, calcitriol, estradiolor estrogens, cortisol, dehydroepiandrosterone, dihydrotestosterone,testosterone, progesterone. Suitable hormones can include, for example,amylin, anti-Müllerian hormone, adiponectin, adrenocorticotropic hormone(or corticotropin), angiotensinogen, angiotensin, antidiuretic hormone(e.g., vasopressin, arginine vasopressin), atrial-natriuretic peptide(e.g., atriopeptin), brain natriuretic peptide, calcitonin,cholecystokinin, corticotropin-releasing hormone, cortistatin,encephalin, endothelin, erythropoietin, follicle-stimulating hormone,galanin, gastric inhibitory polypeptide, gastrin, ghrelin, glucagon,glucagon-like peptide-1, gonadotropin-releasing hormone, growthhormone-releasing hormone, growth hormone, hepcidin, human chorionicgonadotropin, human placental lactogen, inhibin, insulin, insulin-likegrowth factor (or somatomedin), leptin, lipotropin, luteinizing hormone,melanocyte stimulating hormone, motilin, orexin, oxytocin, pancreaticpolypeptide, parathyroid hormone, pituitary adenylate cyclase-activatingpeptide, prolactin, prolactin releasing hormone, relaxin, renin,secretin, somatostatin, thrombopoietin, thyroid-stimulating hormone (orthyrotropin), thyrotropin-releasing hormone, or vasoactive intestinalpeptide.

In some embodiments, the viable cells can be primed, by exposing theviable cells to various stimuli such as electrical or mechanical stress(e.g., fluid flow, substrate stiffness, topography, shear, matrixstretching, compression, torque), deprivation or supplementation of oneor more nutrients (e.g., in medium), modulation of pressure,electromagnetic force, ultrasound, shockwave treatment, irradiation,change in temperature (e.g., temperature shock), pH (e.g., change inacidity or alkalinity) and atmospheric oxygen levels (e.g., hypoxia orhyperoxia) for a predetermined duration of time.

In some embodiments, exposure of viable amniotic membrane to hypoxicconditions (either by controlling atmospheric oxygen levels or addingsubstances to affect oxygen levels in the culture medium) can lead toaccelerated angiogenic differentiation of the resident cells and resultin more rapid healing when used treat, e.g., chronic wounds for thepatient.

In some embodiments, the stimuli (physical or biochemical) can beapplied in a static or dynamic fashion at the priming step. The stimulican also be applied transiently (e.g., seconds or minutes) or for aprolonged period of time (e.g., 30 minutes to an hour, or hours ordays). In some embodiments, the one or more stimuli may also be appliedsimultaneously, in sequence, or individually. The result of applying theprocessing conditions to the viable allogeneic cells will be to primethe cells to achieve an improved and/or more rapid therapeutic effectfollowing transplantation.

Priming of viable cells can produce unexpected and advantageous results.For example, in some embodiments, priming of cells can result incellular expression to produce a desired cell phenotype for use intherapeutic treatments. In some embodiments, priming of cells can resultin faster cell differentiation of immature cells. In some embodiments,priming of cells can result in a greater percentage of immature cellsthat differentiate into target cells. In some embodiments, priming ofcells can result in production of desired ECM. In some embodiments,priming of cells can result in the secretion of biochemical factors thatenhance healing response. In some embodiments, priming of cells canresult in the inhibition of apoptosis and improved cell viability. Insome embodiments, priming of cells can result in the de-differentiationof mature or senescent cells. In some embodiments, priming of cells canpromote or confer immunomodulatory, anti-infective properties,anti-inflammatory properties, and/or anti-scarring properties. In someembodiments, priming of cells can increase mobility of cells. In someembodiments, priming of cells can induce cellular reorganization. Insome embodiments, priming of cells can induce cellular proliferation. Insome embodiments, priming of cells can result in the restoration ofproliferative and functional capacity to senescent cells either in thetissue or at the host site. In some embodiments, priming of cells canresult in the selective elimination of senescent or otherwise undesiredcells in the tissue. In some embodiments, priming of cells can reduce orinhibit innate immunoreactivity or inflammatory responses of tissuesand/or organs.

The effects of cellular priming can be characterized by qualitative andquantifiable aspects of cellular physiology. For example, cellularpriming can be evaluated by characteristics such as time todifferentiation, percentage of differentiated cells, cell viability,cell proliferative activity, metabolic activity, cell morphology,epigenetic marker profile, surface marker expression, gene expressionprofiles, ECM production, secretion of biochemical factors, healing orremodeling in an in vitro or in vivo model, recruitment of host cells todefect site, modification of endogenous cell phenotypes, and/ormodification of cell phenotypes at the host site in response to secretedfactors/produced ECM.

In one example, a sample can be set aside in order to evaluate cellcount and cell viability/biological activity of the tissue usingcommercially available methods, including but not limited to, forexample, metabolic assays, such as involving luciferase, tetrazoliumsalts (e.g., 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT),3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MT S),2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide(XTT), and other water soluble tetrazolium salts (e.g., WST-1, -3, -4,-5, -8, -9, -10, and -11), live-dead assays, ATP assay, CCK-8 assay anddye exclusion assays such as Trypan Blue.

In some embodiments, cell priming or pre-conditioning could also beperformed to counteract the negative effects that traditional processingmethods and the time between recovery and processing would have on theallograft tissue and cells. For example, priming of viable cells canreduce the level of inflammation of the tissue upon implantation,thereby improving transplant acceptance and reducing patient recoverytime. In addition, endogenous pre-conditioned cells could be directed toproduce extracellular matrix or other biochemical factors that would bebeneficial when introduced into the graft site in the patient. Primingcan also reduce risk of infection, reduce risk of graft rejection,and/or reduce level of inflammation.

After priming, grafts containing primed cells could be provided eitherin a fresh or cryopreserved state to a patient. In some embodiments,immunoreactive cells may be removed from the graft while the cells ofinterest remain bound to the tissue. Methods of removal may be differentfor different tissue types. One exemplary method includes rinsing orsoaking the tissue in a chemical solution for immunodepletion (e.g., theProteoPrep® 20 Plasma Immunodepletion Kit from Sigma-Aldrich Co. LLC).

In one example, viable amniotic membrane for enhanced wound healing canbe prepared by the following steps:

(1) Recover fresh amnion;(2) Expose cells to hypoxic conditions for specific duration of time;(3) Cryopreserve viable allograft or offer as fresh tissue; and(4) Prepare/prime amnion cells for angiogenesis.

In another example, viable bone or periosteum for enhanced bone healingcan be prepared by the following steps:

(1) Recover fresh cancellous or periosteum;(2) Expose cells to osteogenic medium for specific duration of time;(3) Cryopreserve viable allograft; and(4) Prepare/prime amnion cells for osteogenesis.

In some embodiments, the cells can be strategically primed to producebeneficial soluble factors (e.g., adipokines, cytokines, growth factors,etc.) and/or extracellular matrix (ECM). In some embodiments, the primedcells contained within a tissue of interest can be at least partiallydecellularized or devitalized prior to implantation to a patient. Thetissue (e.g., allograft) can then be primed for desired incorporationand remodeling, potentially without the need to maintain the livingcells. In some embodiments, this can be used in off-the-shelf graftconfigurations. Without wishing to be bound by theory, one hypothesis isthat the extracellular matrix and/or biochemical factors that the cellsproduced prior to decellularization or devitalization may still affecthost cells following transplantation. Decellularization is the removalof at least some of the endogenous cells and can optionally includekilling of the cells. Any decellularization methods known in the art canbe used. For example, a tissue can be treated by a solution containingdetergents and/or hypotonic saline, to loosen and rinse off at least aportion of the cells in the tissue.

Devitalization is the killing of cells and could occur with or withoutcell removal. Any devitalization methods known in the art can be used inthe preparation of, e.g., cell-free grafts. It aims to remove all orsubstantially all cellular material without adversely affecting thecomposition, mechanical integrity or biologic activity of the remainingECM that carries specific properties. In some embodiments, physicaltreatment (e.g., freeze & thaw cycles, sonication, pressure, andmechanical agitation), enzymatic treatment (e.g., Trypsin) or chemicaltreatment (e.g., sodium deoxycholate, Triton X solutions, alcohol,peroxide, dilute acid) can be used to eliminate the living fraction fromthe primed tissue. Those methods should achieve preservation of the ECMproperties while obtaining an efficient removal of the cellularcomponent.

In certain embodiments, the resident cells can be primed to secretebiochemical factors and then leave behind residual amount of thesefactors which could, in turn, also elicit an enhanced healing responseonce the graft is applied to the patient. Secreted factors can includeECM components such as collagen(s), elastin, hydroxyapatite,proteoglycan, hyaluronan, fibronectin, and laminin. The collagen couldbe any type of collagen. Other factors secreted by primed cells caninclude angiogenic factors, mitogenic factors, osteogenic factors,adipogenic factors, chondrogenic factors, antimicrobial factors, andanti-inflammatory factors, including without limitation BMP, TGF-β, FGF,VEGF, PDGF, EGF, HGF, IGF, IL, β-defensin, insulin, ephrin, GDNF, GDF,NGF, KGF, TNF-α, TGF-α, interferon, EPO, albumin, transferrin, andSDF-la.

In some embodiments, the resident cells can be primed to secretebiochemical agents or biochemically active factors into the mediumsurrounding the tissue. In this manner, a conditioned medium can becreated that may utilized either directly or indirectly for therapeuticpurposes. In some embodiments, the conditioned medium can be injected orapplied to the targeted site within or on the body. In some embodiments,the conditioned medium can be applied to other viable cells (e.g.,allogeneic, autogenic, xenogeneic) in order to stimulate a desiredresponse (e.g., growth, migration, differentiation, de-differentiation,trans-differentiation) from these cells.

For example, to produce the conditioned medium, the resident cells canbe cultured in a basal medium (e.g., DMEM (Dulbecco's modified Eagle'smedium), optionally supplemented with serum, hormones, growth factors,cytokines, antibiotics, trace elements, and other additives. Growthfactors that can be added include, but are not limited to, fibroblastgrowth factors (FGFs), epidermal growth factor (EGF), transforminggrowth factor-β (TGF-β), hepatocyte growth factor (HGF), neuralepidermal growth-factor-like 1 (NELL-1), or oncostatin M. Additives tothe medium may include insulin, transferrin, selenium, glucose,interleukin-6, and histone deacetylase inhibitor such as sodium butyrateor tricostatin A.

In some embodiments, the primed and/or conditioned tissue grafts orcells can be washed, rinsed, or otherwise exposed to solutions to primethe cells in the tissue grafts. In some embodiments, the primed and/orconditioned tissue grafts or cells can be washed, rinsed, or otherwiseexposed to solutions to pre-condition the cells in the tissue grafts. Insome embodiments, the primed and/or conditioned tissue grafts or cellscan be washed, rinsed, or otherwise exposed to solutions to extend ordecrease the viability of the cells resident in the tissue grafts, alterthe cell adhesion characteristics of the cells in the tissue graft orthe tissue graft itself, alter the cell proliferation of the cellsresident in the tissue grafts, maintain the cell phenotype of the cellsresident in the tissue graft, and/or alter the migration capability ofcells resident in the tissue graft.

In some embodiments, the primed and/or conditioned tissue grafts or cellpopulations can be stored cryopreserved (e.g., ≤0° C.), optionally withcryopreservative solutions. In some embodiments, the primed and/orconditioned tissue grafts or cell populations can be stored frozen(e.g., ≤0° C.), optionally with preservative solution. In someembodiments, the primed and/or conditioned tissue grafts or cellpopulations can be stored at refrigerated temperatures (e.g., between 0°C.-10° C.), optionally with preservative solution. In some embodiments,the primed and/or conditioned tissue grafts or cell populations can bestored at room temperatures (e.g., between 10° C.-30° C.), optionallywith preservative solution. In some embodiments, the primed and/orconditioned tissue grafts or cell populations can be stored at elevatedtemperatures (e.g., ≥30° C.), optionally with preservative solution.

In some embodiments, the primed and/or conditioned tissue grafts or cellpopulations that are stored under cryopreservation conditions arederived from fresh or previously frozen versions of one or more of thefollowing: placenta, amnion, chorion, umbilical cord, Wharton's Jelly,bone, periosteum, cartilage, meniscus, spinal disc, muscle, tendon,ligament, adipose, skin, cardiovascular tissue, peritoneum, fascia,nerve, cornea, visceral organ, reproductive tissue, hair follicles,foreskin, and dental tissue.

In some embodiments, the primed and/or conditioned tissue grafts or cellpopulations that are stored under freezing conditions are derived fromfresh or previously frozen versions of one or more of the following:placenta, amnion, chorion, umbilical cord, Wharton's Jelly, bone,periosteum, cartilage, meniscus, spinal disc, muscle, tendon, ligament,adipose, skin, cardiovascular tissue, peritoneum, fascia, nerve, cornea,visceral organ, reproductive tissue, hair follicles, foreskin, anddental tissue.

In some embodiments, the primed and/or conditioned tissue grafts or cellpopulations that are stored in refrigerated conditions are derived fromfresh or previously frozen versions of one or more of the following:placenta, amnion, chorion, umbilical cord, Wharton's Jelly, bone,periosteum, cartilage, meniscus, spinal disc, muscle, tendon, ligament,adipose, skin, cardiovascular tissue, peritoneum, fascia, nerve, cornea,visceral organ, reproductive tissue, hair follicles, foreskin, anddental tissue.

In some embodiments, the primed and/or conditioned tissue grafts or cellpopulations that are stored at room temperature conditions are derivedfrom fresh or previously frozen versions of one or more of thefollowing: placenta, amnion, chorion, umbilical cord, Wharton's Jelly,bone, periosteum, cartilage, meniscus, spinal disc, muscle, tendon,ligament, adipose, skin, cardiovascular tissue, peritoneum, fascia,nerve, cornea, visceral organ, reproductive tissue, hair follicles,foreskin, and dental tissue.

In some embodiments, the primed and/or conditioned tissue grafts or cellpopulations that are stored at elevated temperatures are derived fromfresh or previously frozen versions of one or more of the following:placenta, amnion, chorion, umbilical cord, Wharton's Jelly, bone,periosteum, cartilage, meniscus, spinal disc, muscle, tendon, ligament,adipose, skin, cardiovascular tissue, peritoneum, fascia, nerve, cornea,visceral organ, reproductive tissue, hair follicles, foreskin, anddental tissue.

In some embodiments, the primed and/or conditioned tissue grafts orcells populations are allogenic to the recipient. In some embodiments,the primed and/or conditioned tissue grafts or cells populations areautogenic to the recipient. In some embodiments, the primed and/orconditioned tissue grafts or cells populations are xenogenic to therecipient.

EXAMPLES Example 1—Osteogenic Differentiation of Amnion Tissue afterCryopreservation

In this example, amnion tissue was analyzed to determine if the abilityto undergo osteogenic differentiation can be retained followingcryopreservation. The experiment was conducted according to thefollowing procedure. Each donor was processed and cryopreserved for 1month according to procedures previously outlined. Briefly, the amnionwas separated, washed, and cut into 2×2 cm sheets. Each sheet waspackaged into a separate vial, cryopreserved, and placed into vaporphase liquid nitrogen storage for 1 month. At the 1 month time point, avial from the corresponding donor were removed from the vapor phaseliquid nitrogen storage tank (cryotank) and placed into the 37° C. waterbath to thaw. The cryoprotectant solution was decanted by gentlyinverting the vial. Dextrose solution (5%) was added to the vial and thevial was left without agitation for 5 minutes. After 5 minutes, the vialwas inverted to remove the dextrose solution. The amnion sheet waspicked up from the mesh material with forceps and carefully cut into 4equal pieces approximately 1×1 cm each and each was placed into a wellof a 24 well plate. Complete DMEM/F12 media was added to each of thewells containing amnion tissue. The media in the wells was changed withfresh complete DMEM/F12 media every 3-4 days. After 2 weeks in completeDMEM/F12 media, the media in 3 of the 4 wells was switched to completeosteogenic media and changed with fresh media every 3-4 days, while the4th well was kept in DMEM/F12 media as a negative control.

At each time point (2, 4, and 6 weeks of osteogenic media) one amnionpiece cultured in osteogenic media was collected and placed into a vialof formalin and fixed overnight. The control well's tissue sample wascollected at the 6 week time point. After fixation, the sample wasplaced into 75% ethanol for storage. The amnion samples were analyzedfor Alizarin Red, Von Kossa, and hematoxylin and eosin (H&E) staining.The Alizarin Red and Von Kossa slides were analyzed for indications ofmineralization in the tissue and the H&E slides were analyzed fordifferences in cell presence in the tissue.

The DMEM/F12 Growth Medium used in the study was generated using thefollowing protocol. EGF and bFGF were reconstituted per manufacturer'sinstructions and stored in the −20° C. freezer. DMEM/F12 basal media wassupplemented with 1% PenStrep, 1% Glutamax, and 10% HI-FBS final v/v,then sterile filtered and stored refrigerated in the dark. CompleteDMEM/F12 was made by adding 0.1% EGF and 0.1% bFGF v/v to supplementDEMEM/F12 immediately before use.

Complete Osteogenic Media used in the study was generated using thefollowing protocol. Dexamethasone was dissolved in a small volume ofabsolute ethanol then added to absolute ethanol to a final volume of25.5 mL (10⁻⁴M stock concentration). Ascorbic acid was dissolved in 10mL of MesenCult™ MSC Basal Medium (10 mg/mL stock concentration).Osteogenic Stimulatory Supplements, β-glycerophosphate, dexamethasone,and ascorbic acid were aliquotted per manufacturer's instructions. Thefollowing components were added to 42 mL of Mesenult MSC Basal Medium:7.5 mL osteogenic stimulatory supplements, 5 μL dexamethasone, 250 μLascorbic acid, 175 μL β-glycerophosphate, and 0.5 mL PenStrep. Thecomplete Osteogenic Media was stored refrigerated in the dark.

The amnion tissue samples were cultured in complete DMEM/F12 growthmedia for 2 weeks in order to help the cells recover from the freezingand thawing process. The osteogenic and control time points were startedon the date when the test tissue samples were first switched to completeosteogenic media.

The post-cryo amnion tissue showed positive Alizarin Red and Von Kossastaining as early as 2 weeks in culture with osteogenic media. Thestained portions were relatively small and dispersed throughout thetissue. By 4 weeks, the stained mineralized portions of tissue werelarger and more prevalent throughout the tissue. Week 6 was alsocomparable, with continuous sections of tissue stained positive withAlizarin Red and Von Kossa and some large nodules visible indicatinghighly mineralized areas. In both the case of Alizarin Red and Von Kossastaining, the stains appear to localize more at the surfaces of theamnion sheet than in the interior of the sheet tissue. This isespecially apparent in the Von Kossa staining, as the Alizarin Redstaining forms larger clumps of red positive staining.

Staining the control tissue at 6 weeks showed no Alizarin Red or VonKossa staining in the tissue besides some artifacts of the stainingprocess. Though there is only one control time point, the lack ofpositive staining after 6 weeks of culture implies that earlier timepoints also do not have positive staining for mineralization. Thus itcan be concluded that amnion from this donor was capable of osteogenicdifferentiation when cultured in osteogenic media after cryopreservationand storage in vapor phase liquid nitrogen for 1 month.

FIG. 2A illustrates Alizarin Red and Von Kossa staining of post-cryoamnion tissue of one donor in osteogenic or control culture conditionsat 2, 4, and 6 week time points. Alizarin Red and Von Kossa staining waspresent in all three time points and not in the control tissue after 6weeks in control growth media. The images were obtained at 10×magnification.

The post-cryo amnion tissue from another donor cultured in osteogenicmedia also exhibited positive Alizarin Red and Von Kossa staining at thefirst time point, 2 weeks. The intensity and size of the mineralizednodules indicated by Alizarin Red increases over time, with the 6 weektissue nearly completely mineralized at the epithelial surfaces of thetissue. Likewise, the Von Kossa staining at 6 weeks is ubiquitous andindicates significant mineralization throughout the tissue. The stainsof both the Alizarin Red and Von Kossa are present at nearly all of theouter surfaces of the sheet tissue, though the interior portions oftissue are not all positively stained by Alizarin Red or Von Kossa.

Again, the control tissue at 6 weeks showed no Alizarin Red or Von Kossastaining. The amnion tissue from this donor was demonstrated to becapable of osteogenic differentiation after cryopreservation and storagein vapor phase liquid nitrogen for 1 month.

FIG. 2B shows Alizarin Red and Von Kossa staining of post-cryo amniontissue of another donor in osteogenic or control culture conditions at2, 3, and 6 week time points. Alizarin Red and Von Kossa staining waspresent in all three time points and not in the control tissue after 6weeks in control growth media. The images were obtained at 10×magnification.

FIG. 2C illustrates the H&E staining of post-cryo amnion tissues of twodonors in osteogenic or control culture conditions at all time points.The images were obtained at 40× magnification. The H&E staining of thepost-cryotank amnion sheets showed cells present in the tissue at alltime points except the 2 week time point for one donor. It is unknownwhy no cells are visible at all at this time point, as the correspondingAlizarin Red and Von Kossa slides show positive staining indicative ofmineralization, and later time points show increased staining in thetissue. At 4 and 6 week time points for the osteogenic media culturedsheets, mineralization can be observed in the H&E slides by the darkpurple nodules visible near the tissue surfaces for both donors.

In conclusion, cryopreserved samples of amnion sheets from two donorswere successfully cultured in osteogenic media for 2, 4, and 6 weeks andshown to exhibit mineralization at all time points by positive stainingof Alizarin Red and Von Kossa. Control sheets cultured in completeDMEM/F12 media displayed no positive staining of either Alizarin Red orVon Kossa at 6 weeks. H&E staining showed that cells were still presentin the sheets after 6 weeks in osteogenic media. The positive AlizarinRed and Von Kossa staining indicate that the cryopreserved sheets fromboth donors were capable of undergoing direct tissue osteogenicdifferentiation after storage in vapor phase liquid nitrogen for 1 monthafter thawing and culturing in growth media for 2 weeks to allow thecells within the tissue to recover from the freezing and thawingprocedures.

Example 2—Osteogenic Differentiation of Fresh Amnion and Chorion Tissue

In this example, fresh amnion and chorion tissue was analyzed todetermine if the tissues can undergo direct osteogenic differentiationafter culturing in osteogenic media for 4, 6, or 8 weeks. The experimentwas conducted according to the following procedure.

The fresh amnion and chorion osteogenic differentiation cultures wereinitiated in according to established protocols in Example 1. Briefly,there were two test and one control minced amnion wells for each ofthree time points, and one test minced chorion well for each of threetime points with one control well overall. Pieces of tissue from thechorion control well were planned to be collected at each time point,effectively allowing a control sample for comparison at each time point.

The media in the wells was changed with fresh complete osteogenic media(for test tissue wells) or complete DMEM/F12 media (“growth media” forcontrol tissue wells) every 3-4 days. At each time point (4, 6, and 8weeks of osteogenic media) the corresponding amnion and chorion test andcontrol tissue was collected and fixed in separate vials of formalinovernight. Several minced tissue pieces from the chorion control well'stissue were collected at each time point as representative controltissue. After fixation, the sample was placed into 75% ethanol forstorage. The samples were analyzed for Alizarin Red, Von Kossa, andhematoxylin and eosin (H&E) staining. The Alizarin Red and Von Kossaslides were analyzed for indications of mineralization in the tissue andthe H&E slides were analyzed for differences in cell presence in thetissue. Complete DMEM/F12 and osteogenic media was generated accordingto procedures outlined in Example 1.

By 4 weeks, tissue from one of the amnion minced test wells showedpositive Alizarin Red and Von Kossa staining, while the other test wellhad much weaker staining. Again at 6 weeks, one test well tissue samplehad stronger positive Alizarin Red and Von Kossa staining than tissuefrom the second test well. However, by week 8 both amnion test wellsamples had similarly strong Alizarin Red and Von Kossa staining whilethe control well tissue remained negative.

Overall, osteogenic differentiation is demonstrated in fresh mincedamnion tissue by 8 weeks with some differences in differentiationkinetics. The control wells did not show any significant positivestaining at any time.

FIG. 3A illustrates Alizarin Red staining of fresh minced amnion tissuein osteogenic or control culture conditions at 4, 6, and 8 week timepoints. Positive staining was visible in test tissue at all three timepoints, and not in the control tissue at any time point. Onerepresentative image from each of two test wells shown at each timepoint. The images were obtained at 10× magnification.

FIG. 3B illustrates Von Kossa staining of fresh minced amnion tissue inosteogenic or control culture conditions at 4, 6, and 8 week timepoints. Positive staining was visible in test tissue at all three timepoints, and not in the control tissue at any time point. Onerepresentative image from each of two test wells shown at each timepoint. The images were obtained at 10× magnification.

FIG. 3C illustrates H&E staining of fresh minced amnion tissue inosteogenic or control culture conditions at 4, 6, and 8 week timepoints. One representative image from each of two test wells shown ateach time point at 40× magnification. The H&E staining of the freshminced amnion tissue showed cells present in the tissue at all timepoints, both in the epithelial layer as well as in the stromal layerbelow. There are no major differences in cell numbers or distributionbetween the control and test well tissue samples, nor between differenttime points. However, nodules of H&E staining indicating mineralizationcan be observed in the epithelial layer of tissue cultured in osteogenicmedia. The H&E nodules are further supporting evidence that the mincedtissue in osteogenic media underwent osteogenic differentiation over thecourse of the study.

FIG. 3D illustrates Alizarin Red staining of fresh minced chorion tissuein osteogenic or control culture conditions at 4, 6, and 8 week timepoints. Positive staining was visible in test tissue at all three timepoints and increases over time while control remains negative at alltime points. The images were obtained at 10× magnification.

By 4 weeks, tissue from the test well containing minced chorion tissueshowed strong positive Alizarin Red and Von Kossa staining, permeatingthroughout the tissue interior. The staining was not evenly distributedbut was rather concentrated in some areas and negative in others. Areaswith many cells tended to have strong staining, whereas areas relativelydevoid of cells did not have any positive staining. The relativeintensity and amount of positive Alizarin Red and Von Kossa stainingincreased from the 4 week to 8 week time point. While early stainingshowed small concentrations of positive Alizarin Red or Von Kossa, the 8week had nearly solid red portions of tissue completely stained withAlizarin Red and large swatches of dense Von Kossa staining, indicatingvery strong mineralization in large portions of the tissue. Areas thatare not stained either had few or no cells visible, suggesting that onlyrelatively acellular areas were not mineralized by 8 weeks. The controlwell tissue did not show any significant positive staining at any time.

The intensity and extent of mineralization in the chorion appears to bestronger than that of the amnion, with positive staining that was notlimited to the tissue surface. The chorion can thus be concluded to havesuccessfully undergone osteogenic differentiation in this tissuedifferentiation study.

FIG. 3E illustrates Von Kossa staining of fresh minced chorion tissue inosteogenic or control culture conditions at 4, 6, and 8 week timepoints. Positive staining was visible in test tissue at all three timepoints and increases over time while control remains negative at alltime points. Images were obtained using a 10× objective.

FIG. 3F illustrates H&E staining of fresh minced chorion tissue inosteogenic or control culture conditions at 4, 6, and 8 week timepoints. Images were obtained at 40× magnification. The H&E staining ofthe fresh minced chorion tissue revealed at least two distinct regionsof chorion: a dense layer of matrix with cell distribution similar tothat of the stromal layer in amnion and a looser layer mostly consistingof cells. Over the three time points, the dense layer did not appear tochange significantly in either the osteogenic or control media wells.The looser layer of cells, on the other hand, mineralized in osteogenicmedia over time which was visualized by the nodules of H&E.

In conclusion, the fresh minced tissue samples of amnion and chorionwere successfully cultured in osteogenic media for 4, 6, and 8 weeks andgenerally demonstrated to have mineralization at each time point bypositive staining of Alizarin Red and Von Kossa. Control wells of mincedamnion and chorion cultured in complete DMEM/F12 media demonstrated nopositive staining of either Alizarin Red or Von Kossa at any time point.H&E staining showed that cells were still present in the test andcontrol tissue samples after 8 weeks in osteogenic media. The positiveAlizarin Red and Von Kossa staining indicated that the fresh mincedtissue from both amnion and chorion was capable of undergoing directtissue osteogenic differentiation. There was some variability in theamount of mineralization present between amnion samples over time, butthe chorion tissue was strongly osteogenic and had increasingly intensepositive staining over time.

Example 3—Osteogenic Differentiation Potential of Amnion and ChorionTissue after Cryopreservation

In this example, amnion and chorion tissue was analyzed to determine ifthe tissues can undergo osteogenic differentiation after 1 month ofcryopreservation. The experiment was conducted according to thefollowing procedure.

Tissue from each donor was processed and cryopreserved according toprocedures disclosed herein. Briefly, the amnion and chorion membraneswere separated, washed, and cut into 2×2 cm sheets. Each sheet wasminced and the minced pieces packaged into a separate cryovial,cryopreserved, and placed into liquid nitrogen storage for 1 month. Atthe 1 month time point, the vials were removed from the vapor phaseliquid nitrogen storage tank (cryotank) and placed in a floater into the37° C. water bath to thaw. The cryoprotectant solution was decanted bygently inverting the vial. 5% dextrose solution was added to the vialuntil full and the vial was left without agitation for 5 minutes. After5 minutes, the vial was inverted to remove the dextrose solution and thetissue was transferred into a well plate. The well plate was gentlyshaken by hand to slightly disperse the minced tissue pieces and asterile mesh was placed into each well to keep the pieces from floatingaround.

Complete DMEM/F12 media was added to each of the wells containing amniontissue. The media in the wells was changed with fresh complete DMEM/F12media every 3-4 days. After 2 weeks in DMEM/F12 media, the media in 3 ofthe 4 wells was switched to complete osteogenic media and changed withfresh media every 3-4 days, while the 4th well was kept in DMEM/F12media as a negative control. At each time point (2, 4, and 6 weeks ofosteogenic media) one amnion piece cultured in osteogenic media wascollected and placed into a vial of formalin and fixed overnight. Thetissue sample in the control well was collected at the 6 week timepoint. After fixation, the sample was placed into 75% ethanol forstorage. The amnion samples were analyzed for Alizarin Red, Von Kossa,and hematoxylin and eosin (H&E) staining. The Alizarin Red and Von Kossaslides were analyzed for indications of mineralization in the tissue andthe H&E slides were analyzed for differences in cell presence in thetissue. Complete DMEM/F12 and osteogenic media were generated accordingto procedures outlined in Example 1.

By 2 weeks, positive Alizarin Red and Von Kossa staining were visible inthe amnion tissue, though there was variability between the duplicatetest tissue samples in the intensity of Alizarin Red staining. Overallthe strongest Alizarin Red staining was observed at the last time point,6 weeks, while Von Kossa staining was strong throughout the time points.Both Alizarin Red and Von Kossa stains were mostly constrained to theepithelial layer, with some stromal layer Alizarin Red staining at 6weeks. These observations were corroborated by the H&E staining whichshows mineralized nodules in the 6 week osteogenic media tissue alongthe epithelial layer but less mineralization in the stromal layer. Atall time points and for both the osteogenic and growth media tissue,cells could be observed in both the epithelial layer as well as in thestromal layer of amnion.

Overall, osteogenic differentiation was demonstrated in thepost-cryopreservation (postcryo) minced amnion tissue by 2 weeks with anincrease in stain intensity from weeks 2 to 6. The Von Kossa stainingalso appeared to be more ubiquitous than Alizarin Red, staining nearlythe entirety of the epithelial layer of amnion while Alizarin Redstaining was more local and occurred in nodules rather than stainingpositive in the entire epithelial surface. The control wells did notshow any significant positive staining at any time. The trend ofosteogenic differentiation seemed to be similar to that of fresh mincedamnion, with weaker staining in the beginning that increases over time.However, the postcryo minced amnion had stronger Von Kossa stainingearlier than fresh minced amnion as well as stronger Alizarin Redstaining at 6 weeks though the fresh minced amnion at 8 weeks ultimatelyhas the strongest Alizarin Red staining (the postcryo study ended at 6weeks).

FIG. 4A illustrates Alizarin Red staining of postcryo minced amniontissue in osteogenic or control culture conditions at 4, 6, and 8 weektime points. Positive staining was visible in test tissue at all threetime points, and not in the control tissue at any time point. Onerepresentative image from each of two test wells shown at each timepoint. Images were obtained at 10× magnification. FIG. 4B illustratesVon Kossa staining of postcryo minced amnion tissue in osteogenic orcontrol culture conditions at 2, 4, and 6 week time points. Positivestaining was visible in test tissue at all three time points, and not inthe control tissue at any time point. One representative image from eachof two test wells is shown at each time point. Images were obtained at10× magnification.

FIG. 4C illustrates H&E staining of postcryo minced amnion tissue inosteogenic or control culture conditions at 2, 4, and 6 week timepoints. One representative image from each of two test wells is shown ateach time point. Images were obtained at 40× magnification.

By 2 weeks, positive Alizarin Red and Von Kossa staining were visible inthe chorion tissue. Interestingly, week 4 staining showed significantlyless Alizarin Red and Von Kossa staining, with one of two test tissuesamples having effectively no positive Alizarin Red or Von Kossastaining at all when compared to the corresponding control tissue. Atweek 6, one of the Alizarin Red stains remained negative while the othertest sample stained strongly for Alizarin Red, more so than week 2samples. The Von Kossa stain also increased from week 4 to 6, with bothtest tissue samples showing levels of staining stronger than week 2samples. The H&E stains showed cells present in the chorion throughoutthe time points, with nodules of mineralization observed in the week 4samples. Week 6 had significant mineralization visible, as expected ofthe time point with the strongest Alizarin Red and Von Kossa staining.

Overall, osteogenic differentiation is demonstrated in the postcryominced chorion tissue by 2 weeks with an increase in stain intensityfrom weeks 2 to 6. The control wells did not show any significantpositive staining at any time. Compared to the fresh minced chorion, thestains at each postcryo time point were weaker than the correspondingstains at the fresh time point, with the exception of Von Kossa at 6weeks where the stains were relatively similar. Whereas fresh mincedchorion had noticeably much stronger osteogenic differentiation thanfresh amnion tissue, the difference between the two tissues postcryo wasless pronounced.

FIG. 4D illustrates Alizarin Red staining of fresh minced chorion tissuein osteogenic or control culture conditions at 2, 4, and 6 week timepoints. One representative image from each of two test wells is shown ateach time point. Images were obtained at 10× magnification.

FIG. 4E illustrates Von Kossa staining of fresh minced chorion tissue inosteogenic or control culture conditions at 2, 4, and 6 week timepoints. Positive staining is present at all three time points whilecontrol remains negative at all time points. One representative imagefrom each of two test wells is shown at each time point. Images wereobtained at 10× magnification.

FIG. 4F illustrates H&E staining of fresh minced chorion tissue inosteogenic or control culture conditions at 2, 4, and 6 week timepoints. One representative image from each of two test wells is shown ateach time point. Images were obtained at 40× magnification.

In conclusion, postcryo minced tissue samples of amnion and chorion weresuccessfully cultured in osteogenic media for 2, 4, and 6 weeks anddemonstrated to have mineralization at the first time point by positivestaining of Alizarin Red and Von Kossa. Control wells of minced amnionand chorion cultured in complete DMEM/F12 media exhibited no positivestaining of either Alizarin Red or Von Kossa at any time point. H&Estaining showed that cells were still present in the test and controltissue samples after 6 weeks in media, with mineralized nodules apparentin some of the test H&E slides. The positive Alizarin Red and Von Kossastaining indicate that the postcryo minced tissue from both amnion andchorion was capable of undergoing direct tissue osteogenicdifferentiation. Both the amnion and chorion tissue had weaker stainingcompared to that of fresh tissue though the chorion had a relativelylarger decrease from fresh to postcryo tissue staining of both Alizarinand Von Kossa.

Example 4—Chondrogenic Potential of Amnion Cells

In this example amnion tissues were utilized to investigate thechondrogenic differentiation potential of the cells in variousconfigurations (e.g., minced, sheet). These experiments were conductedaccording to the following procedure.

In one experiment, amnion tissue from a placental donor was minced andtransferred with forceps into a tissue culture (TC)-treated 24 wellplate. There were 3 pieces of minced tissue total, with each piece ofminced tissue placed into a separate well. The planned time point was 4weeks. In a second experiment, minced tissue pieces from a differentdonor amnion were transferred with forceps into 2 TC-treated 48 wellplates. Each well plate had 3 minced tissue pieces each in separatewells. One extra piece of minced tissue was available, and was placedinto one of the well plates in a separate well for a third time point.The planned time points were 2 weeks and 4 weeks, with an extra mincedpiece planned for 6 weeks. For both experiments, cells were fed withcomplete chondrogenic media, replaced every 3-4 days. At each timepoint, each of the corresponding minced tissue pieces were removed fromthe well plates with forceps and placed into separate microcentrifugetubes with 10% neutral buffered formalin (enough to cover the tissue) tofix for 3 days. After 3 days, each minced tissue piece was transferredwith forceps into new separate microcentrifuge tubes with 70% ethanol(enough to cover the tissue) for storage until sent to a histology labfor Safranin O staining. Slides sent from the histology lab wereanalyzed for positive Safranin O staining, a solid red/orange-red color.

Complete Lonza Chondrogenic Media for the experiment was generated usingthe following procedure. First, TGF-β1 was reconstituted permanufacturer's instructions and stored in the −20° C. freezer. Next thecontents of each component in the Chondrogenic SingleQuots kit exceptGA-1000 were added to 185 mL of basal medium per manufacturer'sinstructions. Then 1% PenStep final v/v was substituted for the GA-1000(gentamycin). The supplemented Chondrogenic Media was then storedrefrigerated in the dark. Immediately before chondrogenic medium wasused, TGF-β1 was added to the medium at 0.05% v/v to make completechondrogenic media.

As illustrated in FIG. 5A, from the first experiment, Safranin Ostaining was observed along the periphery of the tissue afterchondrogenic differentiation of minced amnion tissue. The color doesappear to be similar to images of positive Safranin O staining (FIG. 5A,arrows) as reported by Issekutz et al. Immunol Cell Biol. 2003 October;81(5):397-408.

From the second experiment, there appears to be positive Safranin Ostaining at the periphery of the minced amnion tissue followingchondrogenic differentiation, as illustrated in FIG. 5B. There does notseem to be significant differences in the amount of positive Safranin Ostaining between the three time points.

In conclusion, the minced amnion tissue samples from two distinct donorsappeared to have a detectable level of chondrogenic differentiationaround the periphery of the tissue pieces, as suggested by positiveSafranin O staining. Overall, this study demonstrated that amnion tissueis capable of undergoing chondrogenic differentiation.

Example 5—Osteogenic Differentiation Potential of Amnion Cells inVarious Configurations

In this example amnion cells were utilized to investigate the osteogenicdifferentiation potential of the cells in various configurations (e.g.,monolayer, minced, sheet). The experiment was conducted according to thefollowing procedures.

Monolayer Osteogenic Differentiation

First, cells isolated from a donor amnion were plated into 9 wells eachof 3 TC-treated 24 well plates at 10,000 cells/cm2 (20,000 cells perwell) for monolayer osteogenic differentiation. There were 9 seededwells per well plate for n=3 for each of 3 different stains (AlizarinRed, von Kossa, Alkaline Phosphatase (ALP) stains) in triplicate. Theplanned time points were 2 weeks, 4 weeks, and 6 weeks. Amnion cellsisolated from the same donor were plated into 9 wells each of 2TC-treated 24 well plates at 20,000 cells per well. There were 9 seededwells per well plate for n=3 for each of 3 different stains (AlizarinRed, von Kossa, Alkaline Phosphate (ALP) stains) in triplicate. Theplanned time points were 2 weeks and 4 weeks. All plates were fed withcomplete DMEM/F12, replaced every 3-4 days, until confluent. After thecells were confluent, wells were fed with complete osteogenic mediawithout β-glycerophosphate, replaced every 3-4 days until evidence ofcell multilayering was apparent through visual inspection by invertedmicroscope. After evidence of cell multilayering, all wells wereswitched to complete osteogenic media with β-glycerophosphate, replacedevery 3-4 days. At each time point, the corresponding 24 well plate ofcells was fixed and stained with Alizarin Red, von Kossa, and ALP (3wells per stain). Von Kossa Staining Protocol 1 (see below for details)was followed for stains performed initially. Afterwards, Von KossaStaining Protocol 2 (see below for details) was followed to obtain adarker stain color. Alkaline Phosphatase Staining Protocol 1 (see belowfor details) was followed for stains performed initially. Afterwards,Alkaline Phosphatase Staining Protocol 2 (see below for details) wasfollowed.

Next, amnion cells isolated from a donor amnion were plated into 9 wellseach of 3 TC-treated 24 well plates at 7,500 cells/cm2 (15,000 cells perwell) to investigate mono layer osteogenic differentiation. There were 9seeded wells per well plate for n=3 for each of 3 different stains(Alizarin Red, von Kossa, Alkaline Phosphatase (ALP) stains) intriplicate. The planned time points were 1 week, 2 weeks, and 4 weeks.However, due to reagent availability the 1 week time point was actuallyprocessed on day 9. Cells were plated at a lower density in order todecrease cell aggregation at the center of the wells previously evidentafter initial seeding of the amnion cells. All plates were fed withcomplete DMEM/F12, replaced every 3-4 days, until confluent. All wellswere fed with complete osteogenic media without β-glycerophosphate,replaced every 3-4 days, until evidence of cell multilayering wasapparent through visual inspection by inverted microscope. Afterevidence of cell multilayering, all wells were switched to completeosteogenic media with β-glycerophosphate, replaced every 3-4 days. Ateach time point, the corresponding 24 well plate was fixed and stainedwith Alizarin Red, von Kossa, and ALP (3 wells per stain). Von KossaStaining Protocol 2 was followed. Alkaline Phosphatase Staining Protocol2 was followed.

Minced Osteogenic Differentiation

Tissue from a donor amnion was minced and transferred from culture into3 TC-treated 24 well plates. One piece of minced tissue was placed intoeach of 3 wells per plate for n=3. The planned time points were 2 weeks,4 weeks, and 6 weeks. All wells were fed with complete osteogenic mediawith β-glycerophosphate, replaced every 3-4 days. At each time point,the corresponding 24 well plate was set aside for staining. Each mincedtissue piece was placed into a microcentrifuge tube with 10% neutralbuffered formalin for 3 days. After 3 days, each minced tissue piece wastransferred into 70% ethanol for storage until sent to a histology labfor staining.

Sheet Osteogenic Differentiation

Amnion sheets from a placental donor were transferred from culture intoa TC-treated 12 well plate. There were 2 sheets, each in a separatewell. The end points for the experiment were 4 and 8 weeks. Sheets fromanother donor amnion were transferred from culture into a TC-treated 12well plate. There were 2 sheets in separate wells. The planned timepoint was 4 weeks for both sheets. All wells were fed with completeosteogenic media with β-glycerophosphate, replaced every 3-4 days. Ateach time point, the corresponding 12 well plate was set aside forstaining. Each sheet was placed into 10% neutral buffered formalin for 3days. After 3 days, each sheet was transferred into 70% ethanol forstorage until sent to a histology lab for staining.

Fixation and Staining

Tissue fixation was performed according to the following procedure.Media was aspirated from each well. Wells were washed twice with PBS toremove residual media. A 10% neutral buffered formalin was added to eachwell for 15 minutes to fix cells. After fixing, the formalin was removedfrom the wells and each well was washed twice in water.

Alizarin Red staining was performed according to the followingprocedure. A 2% Alizarin Red solution was prepared by mixing 2 gAlizarin Red in 100 mL water. The pH of the solution was adjusted tobetween 4.1 and 4.3 using HCl. Cells were covered in Alizarin Redsolution for 1-2 minutes. Alizarin Red solution was removed and thewells washed with water until excess dye was removed. Calcium deposits(excluding oxalate) were stained orange-red by the Alizarin Red stainand visible under a microscope.

Von Kossa staining was performed, in some embodiments, by the followingVon Kossa Protocol 1. The following solutions were prepared: 5% silvernitrate solution (5 g silver nitrate to 100 mL water), 5% sodiumthiosulfate (Hypo) solution (5 g sodium thiosulfate to 100 mL water), 5%silver nitrate solution was added to each well designated for von Kossauntil the well bottom was covered. The well plates were placed under a15 W incandescent bulb or the biosafety hood UV lamp to stain for 1hour. The silver nitrate solution was removed and the wells were rinsedwith water until residual silver nitrate solution was removed. A 5% Hyposolution was added to each of the wells for 5 minutes at roomtemperature. The wells were then rinsed with water until residual Hyposolution was removed. The Von Kossa stain appeared brown-black and wasvisible under a microscope.

Alternatively, Von Kossa staining was performed by Von Kossa Protocol 2.Fresh 2% silver nitrate solution was prepared (2 g silver nitrate to 100mL water) and used within one week of preparation. A 2% silver nitratesolution was added to each well designated for von Kossa until the wellbottom was covered. The well plates were covered in aluminum foil toincubate in a dark environment for 10 minutes. The aluminum foil wasremoved and the wells were rinsed twice with water. The well plates wereleft in fresh Millipore water and exposed to a 60 W incandescent lightbulb for 15 minutes. Aluminum foil was placed beneath the well plate tohelp reflect light. The silver nitrate solution was removed and thewells were rinsed twice with water. Von Kossa stain appeared brown-blackand was visible under a microscope.

Alkaline Phosphatase staining was performed, in some embodiments, by thefollowing Alkaline Phosphatase Protocol 1. Alkaline dye mixture wasprepared by adding the following solutions in order: 0.2 mL ofFBB-alkaline solution was added to 0.2 sodium nitrite solution andmixed. The mixture was added to 9 mL of water. A 0.2 mL naphthol AS-BIalkaline solution was added to the mixture. The tube containing the dyemixture was wrapped in aluminum foil to protect from light. The prepareddye mixture was added to each well designated for ALP staining until thebottom was covered. The wells were incubated for 15 minutes at roomtemperature. After 15 minutes, the wells were rinsed with water untilexcess dye was removed. Where alkaline phosphatase was expressed, theALP stain appeared as blue-purple patches visible under a microscope.

Alternatively, Alkaline Phosphatase staining was performed by AlkalinePhosphatase Protocol 2. Alkaline dye mixture was prepared by adding thefollowing solutions in order: A0.2 mL of FRV-alkaline solution was addedto 0.2 sodium nitrite solution and mixed. The mixture was allowed to sitfor 2 minutes and then added to 9 mL of water. 0.2 mL naphthol AS-BIalkaline solution was added to the mixture. The tube containing the dyemixture was wrapped in aluminum foil to protect from light. The prepareddye mixture was added to each well designated for ALP staining until thebottom was covered. The well plates were covered with aluminum foil andincubated in the dark for 15 minutes at room temperature. After 15minutes, the wells were rinsed with water until excess dye was removed.Where alkaline phosphatase was expressed, the ALP stain appeared asred-violet patches visible under a microscope.

Amnion Cell Monolayer Osteogenic Differentiation 1

As illustrated in FIG. 6A, Alizarin Red and von Kossa staining increasedfrom 2 weeks through 6 weeks. Alizarin Red stains calcium deposits red,while von Kossa stains calcium phosphate brown-black. The stains suggestincreasing mineralization of the wells by calcium deposition over thecourse of the osteogenic differentiation study.

Amnion Cell Monolayer Osteogenic Differentiation 2

As illustrated in FIG. 6B, Alizarin Red and von Kossa staining increasedfrom 2 weeks to 4 weeks, which suggest increasing mineralization of thewells by calcium deposition. Compared to the Osteogenic Differentiation1 wells, the Osteogenic Differentiation 2 wells followed a similar trendof increased stain and had comparable Alizarin Red staining, but lessVon Kossa staining at the same time points. The Alizarin Red and vonKossa stains were also more diffuse and evenly spread out rather thanconcentrated in patches as in the Osteogenic Differentiation 1 wells.

Amnion Cell Monolayer Osteogenic Differentiation 3

As illustrated by FIG. 6C, calcium deposits were evident by 9 days (1week time point) in differentiation media. Both Alizarin Red and VonKossa staining showed an increasing amount of red and black staining,respectively, from 1 week to 4 weeks. The 2 week Alizarin Red stainingappeared to be comparable to Osteogenic Differentiation 2 wells, but theVon Kossa staining was stronger and more similar to the OsteogenicDifferentiation 1 wells. However, by 4 weeks the Ficoll cells seemed tobe more strongly stained with Alizarin Red and both Alizarin Red and VonKossa stains are comparable to Osteogenic Differentiation 1 wells. TheAlizarin Red and Von Kossa stains were more diffuse and spread out thanOsteogenic Differentiation 1 wells at all time points, and theirdistribution is comparable to Osteogenic Differentiation 2 wells. TheALP staining was as expected, with high levels peaking at 9-14 days(week 1 and week 2) then decreasing over time afterwards as shown at theweek 4 time point. Overall, this experiment demonstrated that isolatingcells by tissue digestion and separation did not negatively impact theirosteogenic differentiation capability, and that osteogenicdifferentiation may be evident as early as within 9 days.

Amnion Sheet Osteogenic Differentiation

As illustrated in FIG. 6D, Sheet 1 appeared to have more Alizarin Redstain than Sheet 2 at 4 weeks and was strongly concentrated within onearea, both along the surface and deep within the tissue bulk. The VonKossa stain was more diffuse for Sheet 1, with staining both within thetissue and along the edges. The Von Kossa staining along the outer layerseemed to be stronger for Sheet 2 than for Sheet 1, with a more solidline of stained calcium phosphate along the outer edge. Overall, thesefindings indicate that cells within amnion sheet tissue retainosteogenic differentiation capability.

Amnion Sheet Osteogenic Differentiation

As shown in FIG. 6E, at 8 weeks, the amnion sheet had mineralizedconsiderably compared to what was observed at 4 weeks. Positive AlizarinRed staining could be observed all along the tissue, with some areas ofstronger staining where several tissue surfaces in close proximity wereexposed. Von Kossa has also increased staining from 4 to 8 weeks, withsome areas strongly positive at 8 weeks while other edges only have athin line of Von Kossa staining.

The 8 week sheet was cut in half during collection and only one half wassent for histology. The other half was kept for 16 weeks to observe theextent of mineralization after a very long period of time in osteogenicmedia. During sheet collection, it was noticed that the sheet appearedto be more rigid than normal amnion. Amnion pieces generally curl up andflop downward when picked up with forceps (FIG. 6F, top panel), yet thissheet could be kept relatively flat and was able to stay upright evenwhen held with forceps horizontally (FIG. 6F, bottom panel).

Minced Amnion Osteogenic Differentiation

As illustrated in FIG. 6G, Alizarin Red and Von Kossa staining could beobserved in the 2 week time points, showing that mineralization hadbegun within 2 weeks of osteogenic induction. At 4 weeks, both theAlizarin Red and the Von Kossa stains permeated throughout the tissue,staining the tissue positive both along the tissue edges as well aswithin the minced tissue itself. This was a significant change from 2weeks where only the edges were positive. This distribution of positivestaining was also similar to the positive staining of the amnion sheetat 4 weeks. At 6 weeks, nearly the entirety of the visible tissue waspositive for Alizarin Red and Von Kossa.

The minced tissue also behaved like the sheet after several weeks inosteogenic media. The tissue did not droop when held horizontally, andwhen forceps were used to push the tissue downward, the minced tissuereturned to the horizontal position after the forceps were removed.

In conclusion, the example demonstrated that amnion-derived cells (fromexplants or tissue digestion) are capable of osteogenic differentiationin as little as 9 days and could mineralize considerably, as shown bythe significant amount of Alizarin Red visible (which stains forcalcium) and was supported by Von Kossa and Alkaline Phosphatasestaining.

The study also showed that the amnion tissue itself may becomemineralized in a variety of configurations (minced, sheet remains ofexplants, minced remains of explants) with varying degrees and rates ofosteogenic differentiation. An interesting observation was the increasedrigidity of tissue after culture in osteogenic media. Both sheet andminced tissue were able to retain their shape and position even aftermanipulation with forceps. Overall, it can be concluded that amnioncells do have potential for osteogenic differentiation.

Example 6—Histology of Osteogenic Differentiation of Amnion Tissue

In this example, histology of osteogenic differentiation of amniontissue sheets were cut into two portions and were compared at 8 week and16 week (4 month) time points. Amnion sheet osteogenic differentiationwas performed per procedures of Example 5.

As illustrated in FIG. 7A, at 8 weeks, the amnion sheet had positiveAlizarin Red staining all along the tissue in the epithelial layer, withsome areas of stronger staining where several tissue surfaces in closeproximity were exposed due to tissue folding. Von Kossa also showedpositive staining along the epithelial layer.

The other half of the sheet was kept in osteogenic media until the 16week time point. During sheet collection, it was noticed that the sheetappeared to be more rigid than normal amnion. The amnion sheet could beslightly molded and kept a curved shape when bent with forceps, asillustrated in FIG. 7B, left panel. The sheet was also able to stayvertical when held with forceps without any drooping, as illustrated inFIG. 7B, right panel.

The Alizarin Red staining was very strongly positive all throughout thetissue with particularly red areas both in the epithelial surface aswell as within the tissue. The stain appeared to be stronger in the 16week time point, with larger areas of red as well as darker red stains.The Von Kossa stain was also evident at 16 weeks along the epithelialsurface of the amnion sheet.

Example 7—Adipogenic and Osteogenic Differentiation Potential of AmnionTissue

In this example amnion tissue was utilized to investigate the adipogenicand osteogenic differentiation potential. The experiment was conductedaccording to the following procedures.

First, 8 mm biopsy punches of amnion tissue from a donor were platedinto 15 wells each of 3 TC-treated 48 well plates for 5 time points. Onewell plate was designated as the control plate, osteogenic plate, andadipogenic plate. The experimental end points were 2, 4, 6, 8, 16 weeks.Osteogenic plates were fed with complete osteogenic media withβ-glycerophosphate, adipogenic plates were fed with complete adipogenicmedia, and control plates were fed with complete growth media, replacedevery 3-4 days. At each time point, three biopsy tissue samples from thecontrol, osteogenic, and adipogenic well plates were placed into 10%neutral buffered formalin for 1 day. After 1 day, each punch wastransferred into 70% ethanol for storage until sent to a histology labfor staining.

DMEM/F12 complete growth media was generated by the following procedure.EGF and bFGF were reconstituted per manufacturer's instructions andstored in the −20° C. freezer. DMEM/F12 basal media was supplementedwith 1% PenStrep, 1% Glutamax, and 10% HI-FBS final v/v, then sterilefiltered and stored refrigerated in the dark. Complete growth media wasmade by adding 0.1% EGF and 0.1% bFGF v/v to supplement DEMEM/F12immediately before use.

Complete adipogenic media was generated by the following procedure. Onebottle of adipogenic supplement was combined with one bottle ofadipogenic basal medium. A volume of 1% PenStrep was added to thecombined components to make complete adipogenic media.

Complete osteogenic media was generated by the following procedure.Osteogenic Stimulatory Supplements, β-glycerophosphate, dexamethasone,and ascorbic acid were aliquotted and stored per manufacturer'sinstructions. The following components were added to 42.5 mL of MesenultMSC Basal Medium: 7.5 mL osteogenic stimulatory supplements,dexamethasone, 2504, ascorbic acid, 1754, β-glycerophosphate, a volumeof 1% PenStrep final v/v was added to the supplemented Osteogenic Media.The complete Osteogenic Media was stored refrigerated in the dark.

As illustrated in FIG. 8A, control amnion tissue samples cultured ingrowth media did not have any positive Alizarin Red staining throughoutthe 5 time points. By 2 weeks, some Alizarin Red staining could beobserved in the tissue samples cultured in osteogenic media at theepithelial surface layer. At week 4 and week 6, the amount of AlizarinRed had increased, and the sizes of the stained nodules were alsolarger. Significant amounts of positive Alizarin Red staining could beobserved at week 8, with some nodules reaching deep into the amnionmesenchymal layers from the epithelial surface layer. This is indicativeof mineralization not only at the surface by amnion epithelial cells,but also in the mesenchymal cell layers of the tissue. By week 16, therewas a thick, continuous layer of Alizarin Red staining along theepithelial surface layer that reached into the tissue.

FIG. 8B illustrates Von Kossa staining of tissue samples cultured inosteogenic media vs. tissue in growth media. Images were obtained at 10×magnification. Similar trends were observed in the tissue samplescultured in osteogenic media and stained with Von Kossa. By week 2, someVon Kossa staining was visible along the epithelial surface layer of theamnion tissue. Weeks 4 and 6 demonstrated an increase in the thicknessof the stain in the tissue, and the Von Kossa stain was more continuousalong the epithelial layer. Week 8 Von Kossa staining showed penetrationof the stain into the deeper layers of the amnion, suggesting that themesenchymal layers had also mineralized to some extent. At week 16,there were regions of the tissue that contained numerous scattered VonKossa stain, which may be a sign of larger mineralized nodules. VonKossa staining was not observed at any of the time points for controltissue samples.

H&E staining was also performed on the test and control tissue samples.By week 6, the control tissue still had many cells along the epithelialsurface layer, as well as mesenchymal cells deeper within the tissue(FIG. 8C, top left panel). By week 16, nearly all the cells in themesenchymal layers were gone and only some stained nuclei were visiblein the epithelial layer (FIG. 8C, top right panel). The later samples ofosteogenic tissue contain nodules of H&E stain in the epithelial layerthat obfuscate any nuclei stained in those regions (FIG. 8C, bottom leftpanel). However, a number of mesenchymal cells can be seen in thetissue. By week 16, the nodules were even larger so it was still notpossible to discern epithelial cells in the osteogenic tissue throughH&E staining, but it was noted that there were similar numbers ofmesenchymal cells (FIG. 8C, bottom right panel) as compared to theosteogenic tissue at week 6. Looking at only the mesenchymal cells, theloss of mesenchymal cells over time in the control tissue but not inosteogenic tissue was anticipated, as the cells were in osteogenicdifferentiation media and thus not expected to be as proliferative ormotile as they would be in growth media.

FIG. 8D illustrates Oil Red O staining of the tissue samples cultured inadipogenic media vs. tissue in growth media. The images were obtained at20× magnification. As illustrated in FIG. 8D, Oil Red O staining wasalso positive at week 2 on onward for adipogenic test tissue samples,although the corresponding control tissue had comparable staining. Fromweek 2 to week 8, there was a slight increase in intensity of the OilRed O staining in adipogenic tissue samples, but the Oil Red O wasalways only at the epithelial surface layer for all tissue samples. FIG.8E illustrates a 40× magnification image of control tissue stained withOil Red O at week 4. Individual stained droplets can be distinguished inthe epithelial layer of the amnion.

In conclusion, fresh amnion tissue samples cultured in osteogenic oradipogenic media for up to 16 weeks were compared to tissue samplescultured in control growth media. Positive staining for Alizarin Red andVon Kossa indicative of osteogenic differentiation appeared at the firsttime point, week 2, for osteogenic test tissue samples and progressivelyincreased in intensity and prevalence both at the epithelial cell layerat the surface of the tissue as well as at the mesenchymal cell layerswithin the tissue until week 16. During that time, it was also notedthat the number of mesenchymal cells within the osteogenic tissuesamples did not decrease significantly over time, but that there was anoticeable decrease in epithelial and mesenchymal cells over time in thecontrol tissue samples.

Example 8—Osteogenic Differentiation Potential of Amnion Tissue after 6Weeks of Hypothermic Storage

In this example, the osteogenic differentiation potential of amniontissue sheets was investigated following 6 weeks of hypothermic storage.The experiment was conducted according to the following procedures.

Tissue differentiation and staining was performed by the followingprocedure. Amnion tissue from one placental donor was separated, washed,and cut into 2×2 cm sheets. Sheets were packaged in groups of 4 into aseparate Kapak per time point with supplemented DMEM/F12 (lacking growthfactors) and stored hypothermically (e.g., in the 4° C. refrigerator).At the 6 week time point, one set of 4 samples set aside for cellculture was retrieved from storage and cut into approximately 1×2 cmsized pieces using a scalpel. The amnion tissue was designated asosteogenic (2 sample pieces) or control (1 sample piece) and each piecewas placed into a separate well of a 24 well plate. A volume of 1 mL ofcomplete osteogenic or DMEM/F12 growth media was added to each of thecorresponding wells. The media in the wells was changed with theappropriate fresh complete media twice a week. After 4 weeks, all fivetissue pieces were collected, placed into separate vials of formalin,and fixed overnight. After fixation, the samples were placed intoseparate vials with 75% ethanol for storage. The amnion samples wereanalyzed for Alizarin Red (osteogenic), von Kossa (osteogenic), andhematoxylin and eosin (H&E) staining (all samples). The control samplewas sectioned multiple times and stained to serve as negative control toboth the osteogenic and the chondrogenic tissue samples. The slides wereanalyzed for positive staining indicative of mineralization (AlizarinRed and von Kossa, osteogenic).

FIG. 9A illustrates Alizarin Red staining, FIG. 9B illustrates Von Kossastaining, and FIG. 9C illustrates H&E staining of hypothermically storedamnion tissue in osteogenic or control culture conditions. The imageswere obtained at 10× magnification. By 4 weeks, positive Alizarin Redand Von Kossa staining were visible in the amnion tissue. Both AlizarinRed and Von Kossa stains were mostly constrained to the epitheliallayer, with some fibroblast layer Alizarin Red staining. This was alsoreflected in the H&E stain for these tissue samples, showing darkmineralized nodules along the epithelial layer. These results indicatedthat the tissue was still functionally capable of osteogenicdifferentiation within 4 weeks even after 6 weeks of refrigeratedstorage.

In addition, the mineralization of the osteogenic tissue samples iscorroborated by the nodules in the corresponding H&E stains. Bydemonstrating osteogenic differentiation over time, this experimentreveals that it may be possible to store amnion tissue at refrigeratedtemperatures for weeks and retain functionality, without the extraeffort needed to cryopreserve the tissue.

In conclusion, the amnion tissue samples stored at refrigeratedtemperature for 6 weeks without media exchanges were tested forosteogenic differentiation potential. The control tissue did not stainat all, indicating lack of cells or structure for unknown reasons.However, compared to previous stains and images from journal articles,the Alizarin Red and von Kossa appear to be positive. This suggests thatthe tissue was functional and underwent osteogenic differentiation evenafter refrigerated storage for 6 weeks. These results demonstrate thepotential for a functional amnion tissue form that can be stored asrefrigerated tissue as opposed to cryopreserved tissue.

Various aspects of the present disclosure may be used alone, incombination, or in a variety of arrangements not specifically discussedin the embodiments described in the foregoing and is therefore notlimited in its application to the details and arrangement of componentsset forth in the foregoing description or illustrated in the drawings.For example, aspects described in one embodiment may be combined in anymanner with aspects described in other embodiments. Also, thephraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for the use of the ordinalterm) to distinguish the claim elements.

Also, the phraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of“including,” “comprising,” or “having,” “containing,” “involving,” andvariations thereof herein, is meant to encompass the items listedthereafter and equivalents thereof as well as additional items.“Consisting essentially of” means inclusion of the items listedthereafter and which is open to unlisted items that do not materiallyaffect the basic and novel properties of the invention.

INCORPORATION BY REFERENCE

All publications, patents and sequence database entries mentioned hereinare hereby incorporated by reference in their entireties as if eachindividual publication or patent was specifically and individuallyindicated to be incorporated by reference.

We claim:
 1. A method for providing an artificially primed tissue graft,comprising: obtaining a tissue containing viable cells from a donor,wherein the viable cells are endogenous to the tissue and remainresident in the tissue; and priming the viable cells with one or morestimuli comprising simulated hypoxia to produce the artificially primedtissue graft which comprises an increased amount of angiogenic growthfactors compared to a non-primed tissue, wherein when used to treat apatient the artificially primed tissue graft provides a benefit comparedto non-primed tissue.
 2. The method of claim 1, wherein the tissue is anallograft, autograft or xenograft tissue.
 3. The method of claim 1,wherein the tissue is obtained from one or more of placenta, amnion,chorion, umbilical cord, Wharton's Jelly, bone, periosteum, cartilage,meniscus, spinal disc, muscle, tendon, ligament, adipose, skin,cardiovascular tissue, peritoneum, fascia, nerve, cornea, visceralorgan, reproductive tissue, hair follicles, foreskin, and dental tissue.4. The method of claim 1, wherein the viable cells are not isolated fromthe tissue and comprise non-terminally differentiated cells and/ordifferentiated cells.
 5. The method of claim 1, wherein the simulatedhypoxia is indued by exposing the tissue and resident endogenous viablecells to a medium ingredient that simulates hypoxia.
 6. The method ofclaim 5, wherein the medium ingredient comprises deferoxamine.
 7. Themethod of claim 1, wherein the benefit comprises one or more of (1)altered cell adhesion, altered cell proliferation, altered cellsurvival, maintenance of cell viability, mainetenance of cell phenotypeand/or altered cell migration; (2) induced cell differentiation,de-differentiation and/or transdifferentiation; (3) production ofextracellular matrix and/or biochemical factors; (4) faster or improvedhealing or remodeling; (5) reduced risk of infection; (6) reduced riskof graft rejection; and (7) reduced level of inflammation.
 8. The methodof claim 9, further comprising cryopreserving the primed tissue graftand grafting the cryopreserved tissue.
 9. The method of claim 9, furthercomprising grafting the primed tissue graft to a recipient withoutcryopreservation.
 10. The method of claim 9, further comprising removingimmunoreactive cells prior to grafting.
 11. The method of claim 9,further comprising devitalizing the primed tissue graft prior tografting.
 12. The method of claim 9, further comprising decellularizingthe primed tissue graft prior to grafting.
 13. The method of claim 13,wherein devitalizing comprises physical treatment (e.g., freeze-and-thawcycles, sonication, pressure, and mechanical agitation), enzymatictreatment (e.g., Trypsin) and/or chemical treatment (e.g., sodiumdeoxycholate, Triton X solutions).
 14. The method of claim 1, furthercomprising grafting the primed tissue graft to the patient, wherein atthe time of grafting, the primed tissue graft contains the viable cells.